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Microdose effects of drugs and chemicals on the enzymes urease, diastase and trypsin
British Homceopathic Journal
July 1987. Vol. 76. pp. 150-157
Microdose effects of drugs and chemicals on the enzymes urease, diastase and trypsin W. M. P E R S S O N MD*
Abstract Twelve medicinal agents and chemicals were assessed for microdose effects on the enzymes urease, diastase and trypsin. Silver nitrate and mercuric chloride had a slight effect on urease; mercuric chloride, sulphur and after them calcium chloride had marked effect on diastase, microdoses of Iris, phosphoric acid and arsenic on trypsin. Gold chloride had only a slight effect on diastase. The remaining preparations--benzoic acid, platinum chloride and insulin--had no effect. A n investigation was made to determine the effect of dynamized dilutions of iodine on starch, and the pH of phosphoric acid dilutions by electrometric methods. It has been empirically shown that colorimetry provides the most sensitive method for assessing microdose effects (dilutions up to 10-1~176
gramme of pharmacological studies may serve as the groundwork for subsequent clinical studies and bring system into the microdose studies carried out by many workers. The first stage in the programme--determination of the effect of microdoses on animal and plant enzymes--seems all the more to the point since on the one hand it represents an attempt to determine the pharmacodynamic in-vitro effect of microdoses and on the other aims to obtain experimental demonstration of drug-enzyme interaction.
Current scientific views concerning the nature of biological processes in living organisms are based on the laws of physical chemistry. Extensive researches in this area have established the outstanding significance of colloids as the basis for all forms of living matter. Views as to the effect various substances have on the organism have therefore undergone considerable changes. It has been found, for instance, that the physical state in which medicinal substances are taken into the organism is a key factor in their therapeutic effect. Apart from the quantitative aspect of medicinal actions scientists are now also considering their qualitative effects, and this significantly extends the range of activity various substances may have on the elements of living protoplasm. The aim of the work presented in this paper was to determine enzyme sensitivity to microdoses of various drugs and chemicals, the intention being to progress to isolated organs and the living organism at a later stage. Such a proTranslationfrom the Germanof a paper entitled 'Die Einwirkung yon Mikrodosen saemtlicherArzneimittelauf Fermente: Urease, Diastase und Trypsin', Archives lnternationales de Pharmacodynarnie et de Thkrapie 1933;46: 249-67. Translator:A. R. Meuss, FIL, MITI. *ChemicalLaboratories BotanicalInstitute Academyof Sciences Leningrad
Urease The first enzyme selected for quantitative determination of the effect of various microdoses was urease. Before proceeding to describe specific experimental methods we should however like to discuss the general conditions to be observed when working with microdoses. All utensils used (flasks, pipettes, test tubes etc.) must be absolutely clean. Before every experiment they are thoroughly washed in water and then rinsed out with alcohol and ether (not dichromate solution). The distilled water used to make up the dilutions and the glass of the microdose containers must be of consistent quality for the whole series of experiments. Every experimental series of two or three flasks or test tubes requires to have a control run
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V o l u m e 76, N u m b e r 3 , July 1 9 8 7
c o n c u r r e n t l y to p r o v i d e c o l o r i m e t r i c d a t a f o r comparison. A d h e r e n c e to t h e a b o v e c o n d i t i o n s a n d m a x i mum accuracy concerning time intervals and e x p e r i m e n t a l t e c h n i q u e will e n s u r e r e l i a b l e results. In every instance results must be carefully and r e p e a t e d l y c h e c k e d . The details of the method are as follows. Urease was obtained from Charbin's soya produced in 1927. This yields about 5% of the enzyme. Using van Slykc's method, ~we proceeded as follows: Carefully ground soya flour was mixed with ten parts of water and left to stand for two hours, stirring at regular intervals. The solution was then decanted and filtered, the filtrate poured into ten times the anrount of anhydrous acetone and left to stand until flocculation precipitation was complete. 2 The acetone was then poured off and the sediment repeatedly washcd with the same acetone using a Buchncr funnel. The residue was dried in a dessicator for 24 hours and the resulting powder carefully stored. Prior to an experiment a quantity of it was dissolved to make a 0.1% aqueous solution and left to stand for 30 minutes, shaking the vcsscl at intervals. The solution was then filtered and the opalcsccnt filtrate used as required, adding one or two drops of toluene. It should bc noted that we deliberately avoided using a phosphate buffer 3 to avoid introducing unnecessary factors into the work with microdoses. Instead, a fresh enzyme solution with the required pH was made up daily. The substrate used was a 1% solution of urca purissima. Undiluted this has a concentration of approx. 0.2 mol. According to Locvrcn it is least liable to fluctuations in pH (7.37.5). 4 From a number of medicinal preparations and chemicals made up under direct observation in the pharmacy of the Leningrad Society of Hom~eopathic Physicians the following were selected: silver nitrate, mercuric chloride and benzoic acid. For the tests, each test tube contained 35cm 3 of solution, 0.5g urea (approx. 5 cm 3 toluene per litrc), 5 cm 3of the microdose (or 5cm 3 water for the controls) and 10cm 3 0.1% urease. 5 The total volume was 50 cm 3, containing 1% of urea. The test tubes were then kept at a thermostatically controlled temperature of 50~ for 24 hours. Mirodose activities wcrc determined by titrating the urea when it had been in solution for 24 hours (ca. 70%) using 1/10 N HCI with methyl orange as indicator. 15 cm 3 of decomposed urea and two or three drops of methyl orange were used per titration. The end point was reached when the solution turned pink, an indication that the urea-derived ammonia had been neutralized. S i l v e r n i t r a t e 6 w a s f o u n d to s h o w t h e h i g h e s t a c t i v i t y , w i t h d i l u t i o n s u p t o a n d i n c l u d i n g 10 -s inhibiting urease activity.7 Table 1 clearly shows the powerful inhibitory e f f e c t o f s i l v e r n i t r a t e in s o l u t i o n s o f u p to 10 -s. B e y o n d t h i s t h e c u r v e g o e s u p a b r u p t l y . A t 10 -'~ i n h i b i t i o n o f t h e r e a c t i o n w a s a b s o l u t e l y nil.
TABLE 1. 35 c m -~ 1 % urea + 5 c m -~AgNO.~ + I0 c m ~0 . 1 % urease solution
Total content n/10 HC1 in c m ~ (") control 10 " 10 " 10 " 10-H~
70.0 0.42 1.50 69.5 69.2
TABLE 2. 35 c m ~ 1 % urea + 5 c m -~ HgC12 + 10 crn .~ 0 . 1 % urease
Quantity n/10 HCl in cm 3 control 10 .4 10 ~' 10 ~''
68.2 0.75 61.5 65.0
The question had arisen as to how far the mixing factor of the reagents concerned affected the action of the silver nitrate solution on urease--there had been reference to this in the literaturcg--and tests were run to assess this. Apart from one minor difference (in 2 cm 3) which was only slightly above the standard variation, no appreciable deviation from the prcviously determined 9 results was found. M e r c u r i c c h l o r i d e i n 10 .6 d i l u t i o n i n h i b i t s u r e a s e a c t i v i t y , as s t a t e d b y J a c o b y a n d Schmidt.~~ W e o b t a i n e d the s a m e results a n d a l s o p a r t i a l i n h i b i t i o n w a s t h e 1 0 l~ d i l u t i o n 9 ( T a b l e 2.) B e n z o i c acid was f o u n d to h a v e no effect on urease activity. To determine optimtim conditions for demonstrating microdose effects on urease activity a series of experiments was run in which conditions were changed as follows. The effect of variations in temperature, time and reagent ratios on urease and microdose activities was assessed. Apart from those already given, no further basic data were obtained. In experiments carried out in strict adherence to the prescribed conditions the effective optimum temperature for urea activity was at approx. 50~ C. In a further extensive study the colorimetric method using Nessler's reagen02 was developed and applied. The ratio of reagents used was microdose: urcase: nrea=3:3:15, with urease and urea concentrations approximately the same as before (0.1% and 1%). The test tubes were kept at 50~ C for 1 hour, treated with Nessler's reagent and assessed for colour in a Dubosk colorimcter. The results were the same as before, however.
152 It may be c o n c l u d e d , t h e r e f o r e , that urease shows highly c o n s i s t e n t reactions to m i c r o d o s e s of the drugs and chemicals u n d e r investigation. This was f u r t h e r c o n f i r m e d by the fact that no variation was n o t e d with c h a n g e s in time, t e m p e r a t u r e or r e a g e n t ratios.
Diastase F u r t h e r investigations to d e t e r m i n e m i c r o d o s e effects on e n z y m e s w e r e then d o n e using diastase. D a t a w e r e insufficient to establish the best m e t h o d of quantitative analysis for such small doses. We t h e r e f o r e set up the highly sensitive polarimetric m e t h o d a n d c o n c u r r e n t l y used the colorimetric m e t h o d of staining starch with iodine (see below). It was f o u n d that t h e iodine reaction gave clear indication of starch dissolving and r e a c h i n g the e r y t h r o d e x t r i n stage w h e r e polarimetry p r o v i d e d n o indication o f the reaction occurring. The colorimetric m e t h o d was t h e r e f o r e c o n s i d e r e d the m o s t suitable and sensitive for our purposes. It also p r o v e d i m p o r t a n t to work u n d e r c o n d i t i o n s that would clearly d e m o n s t r a t e colour variations close to the limit of sensitivity. S u b s e q u e n t colorimetric comparison would clearly s h o w any deviation in the quantitative scale.
Britbh Homoeopathic Journal TABLE 3. 15 c m ~ 1 % starch + 3 c m ~ HgC12 + 3 c m -~ 0 . 2 % diastase
control 10 ~' Ill j5
pH (foil colorimeter
pH electrometric (platinum electrode)
5.9 6.0 5.9
5.86 6.88 6.52
containing m i c r o d o s e s of m e r c u r i c chloride s h o w e d a m a r k e d deviation in p H , to the effect that the reaction was less acid. The results given in Table 3 show that 10 -15 dilutions are still capable of attacking platinum. It may be assumed that higher dilutions, e.g. 10 2o and 10 -25, will also have an effect, and this is further evidence of the' e x t r e m e sensitivity of platinum, and p e r h a p s o t h e r precious metals, to mercuric chloride. It is i n t e n d e d to do f u r t h e r e x p e r i m e n t a l work in this area.
Two samples of Merck diastase were used, one in 0.1% solution, the other in 0.2% solution. Fresh solutions were made up daily. They were left to stand for 30-45 minutes, shaking periodically, and then repeatedly filtered through Schleicher & Schuell 595 filter paper to remove weighablc particles. (Glass wool should not be uscd for filtration as it contains alkaline constituents which inhibit diastase activity.) 1 or 2 drops of toluene were added to the filtrate, which was then ready for use. A 1% starch solution (Amylum solubile) served as the substrate. Microdoses of the following were used: mercuric chloride, sulphur, calcium chloride, gold chloride, platinum chloride and iodine. Reaction mixtures were made up as follows: each test tube contained 15 cm3 1% starch, 3 cm3microdosc and 3 cm3 of 0.1% (subsequently 0.2%) diastase solution. The test tubes were kept at 38 ~ for two hours, with one control tube to each series of two experimental tubes.
Continuing our discussion of the method it should also be noted that the test tubes were placed in the thermostat at regular 10 minute intervals and removed in sequence at the same intervals. After cooling them for three or four minutes in water, 5 cm3 of decomposed starch were taken from each test tube, using a pipette. and transferred to sample tubes, to which 5 cm~of water were added. Prior to estimation l drop of Lugol's solution (1:20) ~3was added to each tube. This resulted in different degrees of substrate coloration. If the controls were kept at the reaction temperature for 90 minutes. observing all the necessary conditions, the colour would be violet; after 150 minutes it would he orange red. In this series of expcrimcnts the tubes were kept at thc reaction temperature for 120 minutes; the addition of iodine resulted in a range of bordeaux red colours, all at the limit of sensitivity. Using a Dubosk colorimeter we then proceeded to make quantitative assessments based on the colour changes (Tables 4 and 5). It should be noted that the figures represent the mean of no fcwcr than ten readings made in three consecutive experiments. The results were consistently positive, with wlriations between individual tubes not exceeding 0.5-0.8 in the colorimeter. This is within the normal range of experimental error.
The electrometrically d e t e r m i n e d p H of all solutions was 5.68-6.08; foil c o l o r i m e t r y gave e x t r e m e s of 5.9-6.1. B o t h series s h o w e d the reaction to be slightly acid, i.e. the glass o f the c o n t a i n e r did not release alkaline haaterial and did not influence the process. A t this point, s o m e incidental o b s e r v a t i o n s m a d e with p H determinations may be of interest. A p l a t i n u m elect r o d e had b e e n used for e l e c t r o m e t r i c p H d e t e r m i n a t i o n , and the c o n t e n t s of test t u b e s
Looking at the Tables it is i m m e d i a t e l y obvious that mercuric chloride and sulphur yielded interesting results. Initial inhibition was followed by activation, this in turn by inhibition, activation and so on. The literature has quite a few references to the effect of minimal doses of mercuric chloride on biochemical processes. A n e x a m p l e are the very convincing results r e p o r t e d by Stassano~4 c o n c e r n i n g the effect of d y n a m i z e d dilutions of m e r c u r y on e n z y m e activity and
Method.
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Volume 76, N u m b e r 3 , July 1987
XABLE4.
15 c m 3 l % starch + 3 cm 3 mercuric chloride + 3 c m ~0 . 1 % diastase
Control
10 6
10-,0
10 ,s
10-20
10 25
10-3o
10-35
10-40
10-45
10 50
10-55
10 6o
20.0
16.6
20.0
24.2
16.7
20.5
25.2
17.4
20.9
16.4
18.4
20.2
18.7
Mercuric chloride
I"ABCE 5. 15 c m s 1 % starch + 3 c m ~ m i c r o d o s e s + 3 c m 3 0 . 2 % diastase
Control
10 6
10 t0
10 15
10-21) 10-25
10 241
10 35
10 -a0
10 -45
10 5o
10-55
10-60
20.0
17.4
20.5
17.6
24,0
17.4
22.5
19.3
20.8
20.1
21.5
21.8
22.2
20.0
22.7
20.5
17.9
20.0
20.0
20.0
20.0
17.5
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
211.0
Sulphur Calcium chloride Gold chloride Platinum chloride
22
9187202122 ~32425 1 ~25
30~35 ~~55 40
~
Effect of microdoses of mercuric chloride on diastase
endocrine function. Richet's w o r k ~5 on the activation of lactic fermentation also merits attention, as does the work of Schade, 16Schulz 17 and others. The figures given in Tables 4 and 5 may be presented in graphic form (Fig. 1 and 2). T w o sinusoidal curves with m a r k e d periodicity are obtained, and these are similar to the graphs produced by K o e n i g is and J u n k e r 19. It has to be emphasized that the mercuric chloride and sulphur w e r e in extremely high dilutions 2~and gave definite m a x i m a (at 10-30 and 10 -2~ respectively). The amplitude of variation decreased with increasing dilution after those maxima, finally disappearing altogether. The fluctuations may be assumed to be due to electro-ionic factors. 21 A t the limits reached with very high dilutions (10 .30 and above) only electrons are probably involved, and this must be a case of electronic 25
23 I0 21 2O 6 /~ 15 22
19 18 17
Fig. 2
10
15
20
25
30
18 17
16 Fig. 1
21 20
25
35 45
V
Effect of microdoses of sulphur on diastase
Fig. 3
Effectof microdoses of calcium chloride on diastase
catalysis (Langmuir22). Figures 1 and 2 d e m o n s trate this clearly. 23 Microdoses of calcium chloride 24 also gave interesting results (Figure 3), causing activation at 10 .6 but inhibiting diastase activity at 10 ~-~, and thus providing clear confirmation of ArndtSchulz's law. Gold chloride is the other chloride to merit attention. Inhibition of diastase activity was only seen at 10 .6. Platinum chloride had no effect whatsoever. Mercuric chloride thus proved the most powerful reagent. This is due to its extreme sensitivity to the biochemical reaction in question. The experimental data given above, obtained under carefully m o n i t o r e d experimental conditions, permit the conclusion that microdoses of the drugs and chemicals in question have a definite effect on biochemical processes. Having established microdose effects on enzymes, i t was decided to assess the effect on o t h e r objects. An example is the reaction between iodine and starch, which was used to assess the effect of microdoses of iodine. The experimental conditions should be such that the reaction goes to the limits of sensitivity. It was felt that it should be possible to use barely noticeable background
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British H o m o e o p a t h i c J o u r m d
TABLE6 . 5 c m 3 1 % starch + 5 c m -~w a t e r + 1 c m 3 m i c r o d o s e Microdoses of iodine
Colorimeter readings
control 10 -r 10-7 10 8 10 `9 10-I~
30.0 26.9 28.7 29.5 30.0 30.0
case.
27
29
30 Fig. 4
less than with a casein substratc. On the other hand Waldschmidt-Lcitz 26 has shown trypsin to have two components: proteinase, and carboxyl-polypepfidase which causes polypeptide degradation. If therefore peptone is used as a substrate, it is clearly the proteinase fraction of the trypsin that is involved in the present
10
Effect of dynamized dilutions of iodine on starch
coloration and detect the slightest degree of darkening due to microdoses of iodine with the aid of a colorimeter. This was achieved as follows. Every test tube contained 5 cm 3 of 1% starch solution, 5 cm 3 of water and 1 cm3 of the microdose (1 cm 3 of water for the control); three drops of Lugol's solution (1 : 100) were added, with the determination carried out immediately afterwards, using a Dubosk colorimeter. The results, based on several rcpctitions, are shown in Table 6. T h e effect o f d y n a m i z e d d i l u t i o n s o f i o d i n e o n s t a r c h is clearly e v i d e n t . Fig. 4 s h o w s t h e g r a d u a l d i m i n u t i o n o f effect. A n e f f e c t w a s d e m o n s t r a b l e in d i l u t i o n s u p to a n d i n c l u d i n g 10 -s. A f t e r this, c o l o u r d i f f e r e n c e s w e r e n o l o n g e r a p p a r e n t . It is n o t i m p o s s i b l e t h a t this m a y p r o v e a useful m e t h o d for the quantitative determination of m i c r o d o s e e f f e c t s o n c h e m i c a l r e a c t i o n s .
Trypsin T h e t h i r d e n z y m e u s e d in t h e s e e x p e r i m e n t a l studies was trypsin. T h e action of the following p r e p a r a t i o n s w a s d e t e r m i n e d : Iris, p h o s p h o r i c acid, a r s e n i c a n d i n s u l i n . The trypsin was obtained from Kahlbaum Co.; it was received in soda solution, 0.1% in both cases. The substrate was a 1% solution of peptone Witte in 0.5% sodium carbonate (NazCO3). For this, we based ourselves on the fact that peptone, a product of proteolytic degradation, easily yields to further decomposition with trypsin. The amount of trypsin required was therefore
The Biuret reaction was used for the estimation, as it is generally used in work with proteolytic enzymes. According to the literature, 27 0.25% is the optimum conccntration for the copper sulphate (CuSO~) solution. As the work progressed it was however found necessary to abandon this particular reaction. One reason was that the pH of trypsin and peptone solutions mixed in the above proportions was 9.1-9.2 (foil colorimcter). Michaelis 28has shown that the optimum pH for trypsin solutions with peptone used as the substratc is between 7.8 and 8.5. An a t t e m p t to reduce the alkalinity of the solutions by reducing the concentration of soda resulted in bluish pink and dirty purple colours rather than pink once a drop of 0.25% CuSO4 had bccn added, and this was a serious obstacle to determination. On the other hand peptone is hydrolyzed in alkalinc solutions once the pH exceeds 9.0 (Waldschmidt-Lcitz). with 50% hydrolysis at pH 9.7. We thercfore changcd to a method given by H6don 29which uses phenolphthalcin. Reagcnt concentrations were as follows: 0.03% trypsin in 0.1% Na2CO3; 1% peptone in 0,2% NaECO3, and a 1% alcoholic solution of phenolphthalein. Using a Michaelis comparator, the pH of a mixture of the above solutions was 8.0-8.2, i.e. within the ideal limits for trypsin. Trypsin and peptone solutions were freshly made up each day, adding a few drops of toluene to each. Each small flask contained 10 cm 3 of 1% peptone solution per 0.2% Na2CO3; 5 cm 3 microdose and 5 cm ' of 0.03 % trypsin solution per 0.1% NazCO3. The flasks were carefully shaken and then kept at a temperature of 38~ for two hours. Each experimental series consisted of two flasks and one trypsin control. It should be noted that the flasks were placed in the thermostatically controlled chamber at regular 10-minute intervals and removed in the same sequence. They were then cooled with water, and 1 or 2 drops of I ~i phcnolphthalein solution were added before the determination was made. Hddon recommends adding the phenolphthalcin prior to incubation. This results in a pink coloration which fades to the same extent to which the peptone is hydrolyzed to polypeptides and amino acids during incubation. This is due to the fact that phenolphthalein only stains solutions of pH 8 and above. Peptone hydrolysis results in successive polypeptide and amino acid formation causing an increase in acidity. Applied in the conditions given above, Hddon's method results in colorations close to the limit of sensitivity. The question we were considering was however whether phenolphthalein itself would show activity in influencing trypsin activity. We therefore carried out a number of experiments and found that the process was faster when the phenolphthalein had been added prior to incubation. Phenolphthalein clearly activates tryp-
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Volume 76, N u m b e r 3 , July 1987
sin, and this activation can bc demonstrated colorimetrically: 50.0 without and 55.5 with phenolphthalein. Our experiments therefore showcd that Hddon's method could bc markedly improved by adding the phenolphthalein after incubation. We were able to establish the activation effect in percentages as well as colorimetric figures as follows. Percentage calculations were based on the colour changes obtained in experimental studies (without trypsin) and in controls, adding 5 cm 3 of 0.1% Na2CO 3in the first case and a corresponding amount of 0.03% trypsin solution in 0.1% Na2CO 3. After two hours incubation phenolphthalcin was added to both and the colour difference determined in a Dubosk colorimeter. The figures obtaincd were 50.0 (experimental study) and 65.0 (with trypsin). Peptone degradation due to trypsin in the given concentration was therefore equivalent to 15.0 in the eolorimeter. Putting this at 100%, it is possible to determine the changes due to microdose effects on trypsin in per cent. As already stated, 1% phenolphthalcin was used to colour the contents of thc tcst flasks after incubation and colour differences were then determined in a Dubosk colorimeter. Tables 7 and 9 give the results. It should be noted that all the figures given in the tables arc thc final results of four consecutive experimental runs, where the results were equally positive, with the figures checked a minimum of twelve times. Maximum variations between consecutive determinations did not exceed 1.0 and were thus in standard experimental error range.
The effect of microdoses of Iris on the reaction is particularly striking. (Table 7) Activation of the process was always followed by an inhibition which was within neutral range, and then by f u r t h e r a c t i v a t i o n . ( F i g . 5) T h e m a x i m u m e f f e c t w a s s e e n a t a d i l u t i o n o f 10 -1~ w i t h t h e a m p l i t u d e
of the fluctuations progressively decreasing with dilution beyond this maximum and finally becoming zero in the region of 10-9~ "l~176 (Intermediate dilutions between those shown in the graph were not tested. It is possible that further variations occurred in between those that were determined.) Following the graph from left to right one notes a steady decrease, with a degree of inhibition seen in the second half (at 10.o5 and 10"s~
The wave form is clearly sinusoidal, as was the case with the action of mercuric chloride and sulphur on diastase. It should be noted that Iris is a plant material for which no specific active principle has so far been determined; the influence on enzyme activity is nevertheless striking. It is possible that Iris contains a 'vitamin-type' fraction that has not yet been identified and may prove specific for trypsin. Detailed investigation of this would no doubt be of interest. We now come to phosphoric acid, a 60% solution of which was used. Two methods were used to determine microdose activity. The first consisted in electrometric p H determination of dynamized dilutions of phosphoric acid based on quinhydrone determination. To achieve maximum accuracy of results, the test tubes used in the determination were first boiled for 5-111 minutes in 20% hydrochloric acid solution and then thoroughly washed out with distilled water. This ensured complete removal of any alkalinity of the glass that might have interfered with the readings.
Table 8 gives the p H values for phosphoric acid in its undiluted form and in dilutions up to
TABLE 7. 10 cm s 1% peptone + 5 cm s Iris + 5 cm s 0.03% trypsin
Iris 10 -35 50.8 10 711 50.0
Control 50.0 % 5.3 % --
10-6 55.1 10 -4{' 54.3 10-75 52.8
% 34,0 % 28.6 % 18.6
10 t~ 57.0 10 -45 50.0 10 a) 4&5
% 46.6 % -o~ lO.O
i0-~s 54.2 10-5o 52.2 10-a5 52.5
% 28.0 % 14.6 % 16.6
10 -2t~" 51.0 10-55 53.8 10-9t) 51.4
% 6.6 % 25.5 % 9.3
10 25 52.7 10 60 50.0 10-95 50.8
% 18.0 % -% 5.3
10-3~J 55.8 10 65 48.9 10-.x) 50.0
% 38.6 % 7,3 % --
57 56 55 54 52 53 51
oo 45
50 49 48
Fig, 5
Effect of microdoses of Iris on trypsin
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Brit&h Homceopathic Journal
the 10 -1~ Fig. 6 d e m o n s t r a t e s the gradual reduction in acidity which is d e p e n d e n t on hydrogen ion dissociation. The angle of the curve rises as the neutral zone (7.0) is approached and changes once the reaction b e c o m e s slightly alkaline. This is a c o m m o n feature for strong and w e a k acids titrated with alkaline solutions. 3~ We used the m e t h o d given above to examine phosphoric acid dilutions, starting from the 10 -6 (Table 9). Two or three m a r k e d fluctuations were found, with the curve gradually dying down between 10 .35 and 10 -45 (quantitatively expressed by the figures 50.0, 51.0 and 50.0). Table 9 also gives the values for arsenic. H e r e the microdose effects were subject to ArndtSchulz's law. A 10 .6 dilution activated trypsin to a marked degree, 10 -15caused inhibition (Fig. 8). These results are entirely analogous to those seen with the microdose effect of calcium chloride on diastase activity (see above).
TABLE8. Ph of dynamized dilutions of phosphoric acid Ph electrometric original substance 10 ~ 10-2 10-3 I0 4 10-5 10 6 10-7 10 s 10-9 tO-~~ aqua dest.
3.35 3.48 3.69 4.03 4.71 5.70 6.59 7.49 7.48 7.41 7.41 6.97
Microdoses of insulin had no effect on trypsin activity. This was determined using dilutions above 10 .3. This agrees with the results reported by Bier 31 who experimented with small doses of 61 60 59 58 57
6
56 55 54 53
5
52
4
51 50
5
20
25
30
48 47 FTg. 8
0
-1 -2 -3 -4 -5 -6 -7 -8 -9 -10 10 10 10 10 10 10 10 10 10 10
Fig. 6 Graphic representation of the figures given in Table 8 54 52
15 5/
45 50
49 48
Fig. 7 Effect of microdoscs of phosphoric acid on trypsin
Effect of microdoses of arsenic on trypsin
insulin in the treatment of diabetes and also saw no effect. It may be assumed that insulin only acts in relatively large doses, curing diabetes by specific trypsin activation. 32 O n the other hand a 10 .5 dilution of insulin does have an effect on lipase, 33 and this increases its range of activity relating to enzymes at least to some extent. Generally speaking a study of the effect of horm o n e microdoses on the latter would be of considerable interest and might be considered a special subject area for biochemical studies. This has also been confirmed by observations m a d e by A b d e r h a l d e n 34 that both trypsin and erepsin
157
V o l u m e 76, N u m b e r 3 , July 1987 TABLE 9. 10 cm ~ 1 % peptone + 5 cm s microdoses + 5 cm 3 0.03% trypsin
Phosphoric acid Arsenic Insulin
Control
10 -6
%
10 -1{}
%
10 -~s
%
10 20
%
10 25
%
10-30
%
50.0 50.0 50.0
53.2 60.6 50.0
21.3 70.6 --
52.3 53.8 50.0
15.3 25.3 --
50.0 47.8 50.0
-15.3 --
51.6 50.0 50.0
10.6 ---
48.9 50.0 50.0
7.3 ---
51.3 50.0 50.0
8.0 ---
are activated by thyroxine in small doses, with larger doses having an inhibitory effect. Small dose effects of thyroxine are thus also subject to Arndt-Schulz's law. It is intended to do further work on the subject. The experimental findings reported in this paper show that microdoses of the drugs and chemicals under investigation have a definite effect on enzyme activities. The extreme sensitivity of the method we evolved for the purpose made it possible to assess the nature of the microdose effect up to and including dilution to 10~~ The results to some extent confirm the effect of 'hom0eopathic' doses on biological objects such as enzymes. Continuing the study of microdose effects on biological objects we consider the next step to be determination of such effects on erepsin. It is also intended to assess the effects of microdoses on isolated organs and finally the whole living organism. T h e w o r k p r e s e n t e d in this p a p e r was d o n e u n d e r direct supervision of Prof. A . S. G i n s b e r g . W e a r e g r e a t l y ind e b t e d to h i m for his valuable advice, f o r this c o n t r i b u t e d m u c h to the o v e r c o m i n g of technical p r o b l e m s a n d the solution of the p r o b l e m s we h a d set ourselves. W e are also m o s t grateful to D r I. E. Z n a m e n s k y w h o assisted us in the w o r k of p H d e t e r m i n a t i o n .
References and notes
1 van Slycke D, Cullen C. J Biol Chem 1914, 19; 1916, 24. 2 Zakowski J. Hoppe Seylers Z Physiol Chem 1931, 202. 3 van Slycke D, Zacharias G , Cullen G. Dtsch Med Wochensehr 1914, Nr. 40. 4 Loevren St. Bioch Zst 1921, 119. 5 Jacoby M. Bioch Zst 1916, 74, 76, 77; 1918, 85, 87. 6 Schmidt E G . J Biol chem 1928, 78. 7 Feigl F. Z s t f a n g e w Chemie 1931, No. 36. 8 The figures given in Tables 1 and 2 represent the sum of two consecutive titrations (15 cm 3 each, i.e. a total of 30 cm3). The latter thus contain 0.3 g of urea, neutralized in form of ammonia with 100 cm 3 of n/10 HC1 (according to Mar-
schall, 0.003 mg of urea is equivalent to 1 cm 3 of n/10
HCI). 9 Sommer I, Myrbaeck K. Hoppe Seylers Z Physiol Chem 1930, 189. 10 ibid. 11 ibid. 12 Pincussen H. Mikromethodik 1930. 13 Wohlgemuth 1. Fermentmethoden 1913; see also H a t a Z. Biochem Zst 1909, 17. 14 Stassano. Comptes rend Soc biol 1905, 58. 15 Richer C. Comptes rend de l'Acad des Sciences 1892, 114. 16 Schade H. (Russian). Physical Chemistry in Medicine 1928. 17 Schulz H. 'Pharmakotherapie'. 18 Koenig K. Z s t f g e s exp Med 1927, 56. 19 Junker N. Biol Zentralblatt 1925, 45. 20 In our view there is a close connection between these results and the instructive work done by Bier et al. on the therapeutic effect of microdoses of sulphur in furunculosis, erysipelas and septic conditions; this furnishes definite proof that sulphur is highly specific for skin diseases or in other words skin diastase. (See Bier A. M M W 1930, Nos. 36 and 38; Bier A, Bumne E. Med Klin 1928 No. g; Beck S. M M W 1930 No. 39). 21 Krawkow N. Zst ges exper Med 1923, 34. 22 J Amer Chem Soc 1916, 38; 1917, 39; 1918, 40. 23 Dilutions between those shown in the graphs were not investigated. It is certainly possible that the above fluctuations recur between the given dilutions (and if the graphs were continued to show even higher dilutions). 24 Keeser E. Arch exper Pathologic u. Pharmakologie 1931, 160. Willstaetter R, Rohdewald M. Hoppe Seylers Z Physiol Chem 1931, 203. 25 Ziegler K, Doerie M. Zstges exper Med 1931, 78. 26 Waldschmidt-Leitz E, Purr A. Ber 1929, 62. Abderhalden E, Schwab E. Fermentforschung 1931, 12. 27 Canals E, Cabanes E. Bull Soc chimie bio11932, XIV, No. 2. 28 Michaelis L, Dawidson H. Biochem Ztschr 1911, 36. Oppenheimer C. Die Fermente und ihre Wirkungen, 1926. 29 H 6 d o n L. Compt rend Soc biol 1923, 88. 30 Mislowitzer E. 'Determination of hydrogen ion concentrations in fluids' (in Russian), 1932. 31 Bier A. M M W 1930 No. 38. 32 Collago I, Dobreff M. Biochem Ztschr 1925, 165. 33 Muehlbock O , Kaufmann C. Bioch Zst 1931, 238. 34 Abderhalden E, Franke K. 'Fermentforschung' 1928, 9.
July 1987. Vol. 76. pp. 150-157
Microdose effects of drugs and chemicals on the enzymes urease, diastase and trypsin W. M. P E R S S O N MD*
Abstract Twelve medicinal agents and chemicals were assessed for microdose effects on the enzymes urease, diastase and trypsin. Silver nitrate and mercuric chloride had a slight effect on urease; mercuric chloride, sulphur and after them calcium chloride had marked effect on diastase, microdoses of Iris, phosphoric acid and arsenic on trypsin. Gold chloride had only a slight effect on diastase. The remaining preparations--benzoic acid, platinum chloride and insulin--had no effect. A n investigation was made to determine the effect of dynamized dilutions of iodine on starch, and the pH of phosphoric acid dilutions by electrometric methods. It has been empirically shown that colorimetry provides the most sensitive method for assessing microdose effects (dilutions up to 10-1~176
gramme of pharmacological studies may serve as the groundwork for subsequent clinical studies and bring system into the microdose studies carried out by many workers. The first stage in the programme--determination of the effect of microdoses on animal and plant enzymes--seems all the more to the point since on the one hand it represents an attempt to determine the pharmacodynamic in-vitro effect of microdoses and on the other aims to obtain experimental demonstration of drug-enzyme interaction.
Current scientific views concerning the nature of biological processes in living organisms are based on the laws of physical chemistry. Extensive researches in this area have established the outstanding significance of colloids as the basis for all forms of living matter. Views as to the effect various substances have on the organism have therefore undergone considerable changes. It has been found, for instance, that the physical state in which medicinal substances are taken into the organism is a key factor in their therapeutic effect. Apart from the quantitative aspect of medicinal actions scientists are now also considering their qualitative effects, and this significantly extends the range of activity various substances may have on the elements of living protoplasm. The aim of the work presented in this paper was to determine enzyme sensitivity to microdoses of various drugs and chemicals, the intention being to progress to isolated organs and the living organism at a later stage. Such a proTranslationfrom the Germanof a paper entitled 'Die Einwirkung yon Mikrodosen saemtlicherArzneimittelauf Fermente: Urease, Diastase und Trypsin', Archives lnternationales de Pharmacodynarnie et de Thkrapie 1933;46: 249-67. Translator:A. R. Meuss, FIL, MITI. *ChemicalLaboratories BotanicalInstitute Academyof Sciences Leningrad
Urease The first enzyme selected for quantitative determination of the effect of various microdoses was urease. Before proceeding to describe specific experimental methods we should however like to discuss the general conditions to be observed when working with microdoses. All utensils used (flasks, pipettes, test tubes etc.) must be absolutely clean. Before every experiment they are thoroughly washed in water and then rinsed out with alcohol and ether (not dichromate solution). The distilled water used to make up the dilutions and the glass of the microdose containers must be of consistent quality for the whole series of experiments. Every experimental series of two or three flasks or test tubes requires to have a control run
150
151
V o l u m e 76, N u m b e r 3 , July 1 9 8 7
c o n c u r r e n t l y to p r o v i d e c o l o r i m e t r i c d a t a f o r comparison. A d h e r e n c e to t h e a b o v e c o n d i t i o n s a n d m a x i mum accuracy concerning time intervals and e x p e r i m e n t a l t e c h n i q u e will e n s u r e r e l i a b l e results. In every instance results must be carefully and r e p e a t e d l y c h e c k e d . The details of the method are as follows. Urease was obtained from Charbin's soya produced in 1927. This yields about 5% of the enzyme. Using van Slykc's method, ~we proceeded as follows: Carefully ground soya flour was mixed with ten parts of water and left to stand for two hours, stirring at regular intervals. The solution was then decanted and filtered, the filtrate poured into ten times the anrount of anhydrous acetone and left to stand until flocculation precipitation was complete. 2 The acetone was then poured off and the sediment repeatedly washcd with the same acetone using a Buchncr funnel. The residue was dried in a dessicator for 24 hours and the resulting powder carefully stored. Prior to an experiment a quantity of it was dissolved to make a 0.1% aqueous solution and left to stand for 30 minutes, shaking the vcsscl at intervals. The solution was then filtered and the opalcsccnt filtrate used as required, adding one or two drops of toluene. It should bc noted that we deliberately avoided using a phosphate buffer 3 to avoid introducing unnecessary factors into the work with microdoses. Instead, a fresh enzyme solution with the required pH was made up daily. The substrate used was a 1% solution of urca purissima. Undiluted this has a concentration of approx. 0.2 mol. According to Locvrcn it is least liable to fluctuations in pH (7.37.5). 4 From a number of medicinal preparations and chemicals made up under direct observation in the pharmacy of the Leningrad Society of Hom~eopathic Physicians the following were selected: silver nitrate, mercuric chloride and benzoic acid. For the tests, each test tube contained 35cm 3 of solution, 0.5g urea (approx. 5 cm 3 toluene per litrc), 5 cm 3of the microdose (or 5cm 3 water for the controls) and 10cm 3 0.1% urease. 5 The total volume was 50 cm 3, containing 1% of urea. The test tubes were then kept at a thermostatically controlled temperature of 50~ for 24 hours. Mirodose activities wcrc determined by titrating the urea when it had been in solution for 24 hours (ca. 70%) using 1/10 N HCI with methyl orange as indicator. 15 cm 3 of decomposed urea and two or three drops of methyl orange were used per titration. The end point was reached when the solution turned pink, an indication that the urea-derived ammonia had been neutralized. S i l v e r n i t r a t e 6 w a s f o u n d to s h o w t h e h i g h e s t a c t i v i t y , w i t h d i l u t i o n s u p t o a n d i n c l u d i n g 10 -s inhibiting urease activity.7 Table 1 clearly shows the powerful inhibitory e f f e c t o f s i l v e r n i t r a t e in s o l u t i o n s o f u p to 10 -s. B e y o n d t h i s t h e c u r v e g o e s u p a b r u p t l y . A t 10 -'~ i n h i b i t i o n o f t h e r e a c t i o n w a s a b s o l u t e l y nil.
TABLE 1. 35 c m -~ 1 % urea + 5 c m -~AgNO.~ + I0 c m ~0 . 1 % urease solution
Total content n/10 HC1 in c m ~ (") control 10 " 10 " 10 " 10-H~
70.0 0.42 1.50 69.5 69.2
TABLE 2. 35 c m ~ 1 % urea + 5 c m -~ HgC12 + 10 crn .~ 0 . 1 % urease
Quantity n/10 HCl in cm 3 control 10 .4 10 ~' 10 ~''
68.2 0.75 61.5 65.0
The question had arisen as to how far the mixing factor of the reagents concerned affected the action of the silver nitrate solution on urease--there had been reference to this in the literaturcg--and tests were run to assess this. Apart from one minor difference (in 2 cm 3) which was only slightly above the standard variation, no appreciable deviation from the prcviously determined 9 results was found. M e r c u r i c c h l o r i d e i n 10 .6 d i l u t i o n i n h i b i t s u r e a s e a c t i v i t y , as s t a t e d b y J a c o b y a n d Schmidt.~~ W e o b t a i n e d the s a m e results a n d a l s o p a r t i a l i n h i b i t i o n w a s t h e 1 0 l~ d i l u t i o n 9 ( T a b l e 2.) B e n z o i c acid was f o u n d to h a v e no effect on urease activity. To determine optimtim conditions for demonstrating microdose effects on urease activity a series of experiments was run in which conditions were changed as follows. The effect of variations in temperature, time and reagent ratios on urease and microdose activities was assessed. Apart from those already given, no further basic data were obtained. In experiments carried out in strict adherence to the prescribed conditions the effective optimum temperature for urea activity was at approx. 50~ C. In a further extensive study the colorimetric method using Nessler's reagen02 was developed and applied. The ratio of reagents used was microdose: urcase: nrea=3:3:15, with urease and urea concentrations approximately the same as before (0.1% and 1%). The test tubes were kept at 50~ C for 1 hour, treated with Nessler's reagent and assessed for colour in a Dubosk colorimcter. The results were the same as before, however.
152 It may be c o n c l u d e d , t h e r e f o r e , that urease shows highly c o n s i s t e n t reactions to m i c r o d o s e s of the drugs and chemicals u n d e r investigation. This was f u r t h e r c o n f i r m e d by the fact that no variation was n o t e d with c h a n g e s in time, t e m p e r a t u r e or r e a g e n t ratios.
Diastase F u r t h e r investigations to d e t e r m i n e m i c r o d o s e effects on e n z y m e s w e r e then d o n e using diastase. D a t a w e r e insufficient to establish the best m e t h o d of quantitative analysis for such small doses. We t h e r e f o r e set up the highly sensitive polarimetric m e t h o d a n d c o n c u r r e n t l y used the colorimetric m e t h o d of staining starch with iodine (see below). It was f o u n d that t h e iodine reaction gave clear indication of starch dissolving and r e a c h i n g the e r y t h r o d e x t r i n stage w h e r e polarimetry p r o v i d e d n o indication o f the reaction occurring. The colorimetric m e t h o d was t h e r e f o r e c o n s i d e r e d the m o s t suitable and sensitive for our purposes. It also p r o v e d i m p o r t a n t to work u n d e r c o n d i t i o n s that would clearly d e m o n s t r a t e colour variations close to the limit of sensitivity. S u b s e q u e n t colorimetric comparison would clearly s h o w any deviation in the quantitative scale.
Britbh Homoeopathic Journal TABLE 3. 15 c m ~ 1 % starch + 3 c m ~ HgC12 + 3 c m -~ 0 . 2 % diastase
control 10 ~' Ill j5
pH (foil colorimeter
pH electrometric (platinum electrode)
5.9 6.0 5.9
5.86 6.88 6.52
containing m i c r o d o s e s of m e r c u r i c chloride s h o w e d a m a r k e d deviation in p H , to the effect that the reaction was less acid. The results given in Table 3 show that 10 -15 dilutions are still capable of attacking platinum. It may be assumed that higher dilutions, e.g. 10 2o and 10 -25, will also have an effect, and this is further evidence of the' e x t r e m e sensitivity of platinum, and p e r h a p s o t h e r precious metals, to mercuric chloride. It is i n t e n d e d to do f u r t h e r e x p e r i m e n t a l work in this area.
Two samples of Merck diastase were used, one in 0.1% solution, the other in 0.2% solution. Fresh solutions were made up daily. They were left to stand for 30-45 minutes, shaking periodically, and then repeatedly filtered through Schleicher & Schuell 595 filter paper to remove weighablc particles. (Glass wool should not be uscd for filtration as it contains alkaline constituents which inhibit diastase activity.) 1 or 2 drops of toluene were added to the filtrate, which was then ready for use. A 1% starch solution (Amylum solubile) served as the substrate. Microdoses of the following were used: mercuric chloride, sulphur, calcium chloride, gold chloride, platinum chloride and iodine. Reaction mixtures were made up as follows: each test tube contained 15 cm3 1% starch, 3 cm3microdosc and 3 cm3 of 0.1% (subsequently 0.2%) diastase solution. The test tubes were kept at 38 ~ for two hours, with one control tube to each series of two experimental tubes.
Continuing our discussion of the method it should also be noted that the test tubes were placed in the thermostat at regular 10 minute intervals and removed in sequence at the same intervals. After cooling them for three or four minutes in water, 5 cm3 of decomposed starch were taken from each test tube, using a pipette. and transferred to sample tubes, to which 5 cm~of water were added. Prior to estimation l drop of Lugol's solution (1:20) ~3was added to each tube. This resulted in different degrees of substrate coloration. If the controls were kept at the reaction temperature for 90 minutes. observing all the necessary conditions, the colour would be violet; after 150 minutes it would he orange red. In this series of expcrimcnts the tubes were kept at thc reaction temperature for 120 minutes; the addition of iodine resulted in a range of bordeaux red colours, all at the limit of sensitivity. Using a Dubosk colorimeter we then proceeded to make quantitative assessments based on the colour changes (Tables 4 and 5). It should be noted that the figures represent the mean of no fcwcr than ten readings made in three consecutive experiments. The results were consistently positive, with wlriations between individual tubes not exceeding 0.5-0.8 in the colorimeter. This is within the normal range of experimental error.
The electrometrically d e t e r m i n e d p H of all solutions was 5.68-6.08; foil c o l o r i m e t r y gave e x t r e m e s of 5.9-6.1. B o t h series s h o w e d the reaction to be slightly acid, i.e. the glass o f the c o n t a i n e r did not release alkaline haaterial and did not influence the process. A t this point, s o m e incidental o b s e r v a t i o n s m a d e with p H determinations may be of interest. A p l a t i n u m elect r o d e had b e e n used for e l e c t r o m e t r i c p H d e t e r m i n a t i o n , and the c o n t e n t s of test t u b e s
Looking at the Tables it is i m m e d i a t e l y obvious that mercuric chloride and sulphur yielded interesting results. Initial inhibition was followed by activation, this in turn by inhibition, activation and so on. The literature has quite a few references to the effect of minimal doses of mercuric chloride on biochemical processes. A n e x a m p l e are the very convincing results r e p o r t e d by Stassano~4 c o n c e r n i n g the effect of d y n a m i z e d dilutions of m e r c u r y on e n z y m e activity and
Method.
153
Volume 76, N u m b e r 3 , July 1987
XABLE4.
15 c m 3 l % starch + 3 cm 3 mercuric chloride + 3 c m ~0 . 1 % diastase
Control
10 6
10-,0
10 ,s
10-20
10 25
10-3o
10-35
10-40
10-45
10 50
10-55
10 6o
20.0
16.6
20.0
24.2
16.7
20.5
25.2
17.4
20.9
16.4
18.4
20.2
18.7
Mercuric chloride
I"ABCE 5. 15 c m s 1 % starch + 3 c m ~ m i c r o d o s e s + 3 c m 3 0 . 2 % diastase
Control
10 6
10 t0
10 15
10-21) 10-25
10 241
10 35
10 -a0
10 -45
10 5o
10-55
10-60
20.0
17.4
20.5
17.6
24,0
17.4
22.5
19.3
20.8
20.1
21.5
21.8
22.2
20.0
22.7
20.5
17.9
20.0
20.0
20.0
20.0
17.5
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
20.0
211.0
Sulphur Calcium chloride Gold chloride Platinum chloride
22
9187202122 ~32425 1 ~25
30~35 ~~55 40
~
Effect of microdoses of mercuric chloride on diastase
endocrine function. Richet's w o r k ~5 on the activation of lactic fermentation also merits attention, as does the work of Schade, 16Schulz 17 and others. The figures given in Tables 4 and 5 may be presented in graphic form (Fig. 1 and 2). T w o sinusoidal curves with m a r k e d periodicity are obtained, and these are similar to the graphs produced by K o e n i g is and J u n k e r 19. It has to be emphasized that the mercuric chloride and sulphur w e r e in extremely high dilutions 2~and gave definite m a x i m a (at 10-30 and 10 -2~ respectively). The amplitude of variation decreased with increasing dilution after those maxima, finally disappearing altogether. The fluctuations may be assumed to be due to electro-ionic factors. 21 A t the limits reached with very high dilutions (10 .30 and above) only electrons are probably involved, and this must be a case of electronic 25
23 I0 21 2O 6 /~ 15 22
19 18 17
Fig. 2
10
15
20
25
30
18 17
16 Fig. 1
21 20
25
35 45
V
Effect of microdoses of sulphur on diastase
Fig. 3
Effectof microdoses of calcium chloride on diastase
catalysis (Langmuir22). Figures 1 and 2 d e m o n s trate this clearly. 23 Microdoses of calcium chloride 24 also gave interesting results (Figure 3), causing activation at 10 .6 but inhibiting diastase activity at 10 ~-~, and thus providing clear confirmation of ArndtSchulz's law. Gold chloride is the other chloride to merit attention. Inhibition of diastase activity was only seen at 10 .6. Platinum chloride had no effect whatsoever. Mercuric chloride thus proved the most powerful reagent. This is due to its extreme sensitivity to the biochemical reaction in question. The experimental data given above, obtained under carefully m o n i t o r e d experimental conditions, permit the conclusion that microdoses of the drugs and chemicals in question have a definite effect on biochemical processes. Having established microdose effects on enzymes, i t was decided to assess the effect on o t h e r objects. An example is the reaction between iodine and starch, which was used to assess the effect of microdoses of iodine. The experimental conditions should be such that the reaction goes to the limits of sensitivity. It was felt that it should be possible to use barely noticeable background
154
British H o m o e o p a t h i c J o u r m d
TABLE6 . 5 c m 3 1 % starch + 5 c m -~w a t e r + 1 c m 3 m i c r o d o s e Microdoses of iodine
Colorimeter readings
control 10 -r 10-7 10 8 10 `9 10-I~
30.0 26.9 28.7 29.5 30.0 30.0
case.
27
29
30 Fig. 4
less than with a casein substratc. On the other hand Waldschmidt-Lcitz 26 has shown trypsin to have two components: proteinase, and carboxyl-polypepfidase which causes polypeptide degradation. If therefore peptone is used as a substrate, it is clearly the proteinase fraction of the trypsin that is involved in the present
10
Effect of dynamized dilutions of iodine on starch
coloration and detect the slightest degree of darkening due to microdoses of iodine with the aid of a colorimeter. This was achieved as follows. Every test tube contained 5 cm 3 of 1% starch solution, 5 cm 3 of water and 1 cm3 of the microdose (1 cm 3 of water for the control); three drops of Lugol's solution (1 : 100) were added, with the determination carried out immediately afterwards, using a Dubosk colorimeter. The results, based on several rcpctitions, are shown in Table 6. T h e effect o f d y n a m i z e d d i l u t i o n s o f i o d i n e o n s t a r c h is clearly e v i d e n t . Fig. 4 s h o w s t h e g r a d u a l d i m i n u t i o n o f effect. A n e f f e c t w a s d e m o n s t r a b l e in d i l u t i o n s u p to a n d i n c l u d i n g 10 -s. A f t e r this, c o l o u r d i f f e r e n c e s w e r e n o l o n g e r a p p a r e n t . It is n o t i m p o s s i b l e t h a t this m a y p r o v e a useful m e t h o d for the quantitative determination of m i c r o d o s e e f f e c t s o n c h e m i c a l r e a c t i o n s .
Trypsin T h e t h i r d e n z y m e u s e d in t h e s e e x p e r i m e n t a l studies was trypsin. T h e action of the following p r e p a r a t i o n s w a s d e t e r m i n e d : Iris, p h o s p h o r i c acid, a r s e n i c a n d i n s u l i n . The trypsin was obtained from Kahlbaum Co.; it was received in soda solution, 0.1% in both cases. The substrate was a 1% solution of peptone Witte in 0.5% sodium carbonate (NazCO3). For this, we based ourselves on the fact that peptone, a product of proteolytic degradation, easily yields to further decomposition with trypsin. The amount of trypsin required was therefore
The Biuret reaction was used for the estimation, as it is generally used in work with proteolytic enzymes. According to the literature, 27 0.25% is the optimum conccntration for the copper sulphate (CuSO~) solution. As the work progressed it was however found necessary to abandon this particular reaction. One reason was that the pH of trypsin and peptone solutions mixed in the above proportions was 9.1-9.2 (foil colorimcter). Michaelis 28has shown that the optimum pH for trypsin solutions with peptone used as the substratc is between 7.8 and 8.5. An a t t e m p t to reduce the alkalinity of the solutions by reducing the concentration of soda resulted in bluish pink and dirty purple colours rather than pink once a drop of 0.25% CuSO4 had bccn added, and this was a serious obstacle to determination. On the other hand peptone is hydrolyzed in alkalinc solutions once the pH exceeds 9.0 (Waldschmidt-Lcitz). with 50% hydrolysis at pH 9.7. We thercfore changcd to a method given by H6don 29which uses phenolphthalcin. Reagcnt concentrations were as follows: 0.03% trypsin in 0.1% Na2CO3; 1% peptone in 0,2% NaECO3, and a 1% alcoholic solution of phenolphthalein. Using a Michaelis comparator, the pH of a mixture of the above solutions was 8.0-8.2, i.e. within the ideal limits for trypsin. Trypsin and peptone solutions were freshly made up each day, adding a few drops of toluene to each. Each small flask contained 10 cm 3 of 1% peptone solution per 0.2% Na2CO3; 5 cm 3 microdose and 5 cm ' of 0.03 % trypsin solution per 0.1% NazCO3. The flasks were carefully shaken and then kept at a temperature of 38~ for two hours. Each experimental series consisted of two flasks and one trypsin control. It should be noted that the flasks were placed in the thermostatically controlled chamber at regular 10-minute intervals and removed in the same sequence. They were then cooled with water, and 1 or 2 drops of I ~i phcnolphthalein solution were added before the determination was made. Hddon recommends adding the phenolphthalcin prior to incubation. This results in a pink coloration which fades to the same extent to which the peptone is hydrolyzed to polypeptides and amino acids during incubation. This is due to the fact that phenolphthalein only stains solutions of pH 8 and above. Peptone hydrolysis results in successive polypeptide and amino acid formation causing an increase in acidity. Applied in the conditions given above, Hddon's method results in colorations close to the limit of sensitivity. The question we were considering was however whether phenolphthalein itself would show activity in influencing trypsin activity. We therefore carried out a number of experiments and found that the process was faster when the phenolphthalein had been added prior to incubation. Phenolphthalein clearly activates tryp-
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Volume 76, N u m b e r 3 , July 1987
sin, and this activation can bc demonstrated colorimetrically: 50.0 without and 55.5 with phenolphthalein. Our experiments therefore showcd that Hddon's method could bc markedly improved by adding the phenolphthalein after incubation. We were able to establish the activation effect in percentages as well as colorimetric figures as follows. Percentage calculations were based on the colour changes obtained in experimental studies (without trypsin) and in controls, adding 5 cm 3 of 0.1% Na2CO 3in the first case and a corresponding amount of 0.03% trypsin solution in 0.1% Na2CO 3. After two hours incubation phenolphthalcin was added to both and the colour difference determined in a Dubosk colorimeter. The figures obtaincd were 50.0 (experimental study) and 65.0 (with trypsin). Peptone degradation due to trypsin in the given concentration was therefore equivalent to 15.0 in the eolorimeter. Putting this at 100%, it is possible to determine the changes due to microdose effects on trypsin in per cent. As already stated, 1% phenolphthalcin was used to colour the contents of thc tcst flasks after incubation and colour differences were then determined in a Dubosk colorimeter. Tables 7 and 9 give the results. It should be noted that all the figures given in the tables arc thc final results of four consecutive experimental runs, where the results were equally positive, with the figures checked a minimum of twelve times. Maximum variations between consecutive determinations did not exceed 1.0 and were thus in standard experimental error range.
The effect of microdoses of Iris on the reaction is particularly striking. (Table 7) Activation of the process was always followed by an inhibition which was within neutral range, and then by f u r t h e r a c t i v a t i o n . ( F i g . 5) T h e m a x i m u m e f f e c t w a s s e e n a t a d i l u t i o n o f 10 -1~ w i t h t h e a m p l i t u d e
of the fluctuations progressively decreasing with dilution beyond this maximum and finally becoming zero in the region of 10-9~ "l~176 (Intermediate dilutions between those shown in the graph were not tested. It is possible that further variations occurred in between those that were determined.) Following the graph from left to right one notes a steady decrease, with a degree of inhibition seen in the second half (at 10.o5 and 10"s~
The wave form is clearly sinusoidal, as was the case with the action of mercuric chloride and sulphur on diastase. It should be noted that Iris is a plant material for which no specific active principle has so far been determined; the influence on enzyme activity is nevertheless striking. It is possible that Iris contains a 'vitamin-type' fraction that has not yet been identified and may prove specific for trypsin. Detailed investigation of this would no doubt be of interest. We now come to phosphoric acid, a 60% solution of which was used. Two methods were used to determine microdose activity. The first consisted in electrometric p H determination of dynamized dilutions of phosphoric acid based on quinhydrone determination. To achieve maximum accuracy of results, the test tubes used in the determination were first boiled for 5-111 minutes in 20% hydrochloric acid solution and then thoroughly washed out with distilled water. This ensured complete removal of any alkalinity of the glass that might have interfered with the readings.
Table 8 gives the p H values for phosphoric acid in its undiluted form and in dilutions up to
TABLE 7. 10 cm s 1% peptone + 5 cm s Iris + 5 cm s 0.03% trypsin
Iris 10 -35 50.8 10 711 50.0
Control 50.0 % 5.3 % --
10-6 55.1 10 -4{' 54.3 10-75 52.8
% 34,0 % 28.6 % 18.6
10 t~ 57.0 10 -45 50.0 10 a) 4&5
% 46.6 % -o~ lO.O
i0-~s 54.2 10-5o 52.2 10-a5 52.5
% 28.0 % 14.6 % 16.6
10 -2t~" 51.0 10-55 53.8 10-9t) 51.4
% 6.6 % 25.5 % 9.3
10 25 52.7 10 60 50.0 10-95 50.8
% 18.0 % -% 5.3
10-3~J 55.8 10 65 48.9 10-.x) 50.0
% 38.6 % 7,3 % --
57 56 55 54 52 53 51
oo 45
50 49 48
Fig, 5
Effect of microdoses of Iris on trypsin
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Brit&h Homceopathic Journal
the 10 -1~ Fig. 6 d e m o n s t r a t e s the gradual reduction in acidity which is d e p e n d e n t on hydrogen ion dissociation. The angle of the curve rises as the neutral zone (7.0) is approached and changes once the reaction b e c o m e s slightly alkaline. This is a c o m m o n feature for strong and w e a k acids titrated with alkaline solutions. 3~ We used the m e t h o d given above to examine phosphoric acid dilutions, starting from the 10 -6 (Table 9). Two or three m a r k e d fluctuations were found, with the curve gradually dying down between 10 .35 and 10 -45 (quantitatively expressed by the figures 50.0, 51.0 and 50.0). Table 9 also gives the values for arsenic. H e r e the microdose effects were subject to ArndtSchulz's law. A 10 .6 dilution activated trypsin to a marked degree, 10 -15caused inhibition (Fig. 8). These results are entirely analogous to those seen with the microdose effect of calcium chloride on diastase activity (see above).
TABLE8. Ph of dynamized dilutions of phosphoric acid Ph electrometric original substance 10 ~ 10-2 10-3 I0 4 10-5 10 6 10-7 10 s 10-9 tO-~~ aqua dest.
3.35 3.48 3.69 4.03 4.71 5.70 6.59 7.49 7.48 7.41 7.41 6.97
Microdoses of insulin had no effect on trypsin activity. This was determined using dilutions above 10 .3. This agrees with the results reported by Bier 31 who experimented with small doses of 61 60 59 58 57
6
56 55 54 53
5
52
4
51 50
5
20
25
30
48 47 FTg. 8
0
-1 -2 -3 -4 -5 -6 -7 -8 -9 -10 10 10 10 10 10 10 10 10 10 10
Fig. 6 Graphic representation of the figures given in Table 8 54 52
15 5/
45 50
49 48
Fig. 7 Effect of microdoscs of phosphoric acid on trypsin
Effect of microdoses of arsenic on trypsin
insulin in the treatment of diabetes and also saw no effect. It may be assumed that insulin only acts in relatively large doses, curing diabetes by specific trypsin activation. 32 O n the other hand a 10 .5 dilution of insulin does have an effect on lipase, 33 and this increases its range of activity relating to enzymes at least to some extent. Generally speaking a study of the effect of horm o n e microdoses on the latter would be of considerable interest and might be considered a special subject area for biochemical studies. This has also been confirmed by observations m a d e by A b d e r h a l d e n 34 that both trypsin and erepsin
157
V o l u m e 76, N u m b e r 3 , July 1987 TABLE 9. 10 cm ~ 1 % peptone + 5 cm s microdoses + 5 cm 3 0.03% trypsin
Phosphoric acid Arsenic Insulin
Control
10 -6
%
10 -1{}
%
10 -~s
%
10 20
%
10 25
%
10-30
%
50.0 50.0 50.0
53.2 60.6 50.0
21.3 70.6 --
52.3 53.8 50.0
15.3 25.3 --
50.0 47.8 50.0
-15.3 --
51.6 50.0 50.0
10.6 ---
48.9 50.0 50.0
7.3 ---
51.3 50.0 50.0
8.0 ---
are activated by thyroxine in small doses, with larger doses having an inhibitory effect. Small dose effects of thyroxine are thus also subject to Arndt-Schulz's law. It is intended to do further work on the subject. The experimental findings reported in this paper show that microdoses of the drugs and chemicals under investigation have a definite effect on enzyme activities. The extreme sensitivity of the method we evolved for the purpose made it possible to assess the nature of the microdose effect up to and including dilution to 10~~ The results to some extent confirm the effect of 'hom0eopathic' doses on biological objects such as enzymes. Continuing the study of microdose effects on biological objects we consider the next step to be determination of such effects on erepsin. It is also intended to assess the effects of microdoses on isolated organs and finally the whole living organism. T h e w o r k p r e s e n t e d in this p a p e r was d o n e u n d e r direct supervision of Prof. A . S. G i n s b e r g . W e a r e g r e a t l y ind e b t e d to h i m for his valuable advice, f o r this c o n t r i b u t e d m u c h to the o v e r c o m i n g of technical p r o b l e m s a n d the solution of the p r o b l e m s we h a d set ourselves. W e are also m o s t grateful to D r I. E. Z n a m e n s k y w h o assisted us in the w o r k of p H d e t e r m i n a t i o n .
References and notes
1 van Slycke D, Cullen C. J Biol Chem 1914, 19; 1916, 24. 2 Zakowski J. Hoppe Seylers Z Physiol Chem 1931, 202. 3 van Slycke D, Zacharias G , Cullen G. Dtsch Med Wochensehr 1914, Nr. 40. 4 Loevren St. Bioch Zst 1921, 119. 5 Jacoby M. Bioch Zst 1916, 74, 76, 77; 1918, 85, 87. 6 Schmidt E G . J Biol chem 1928, 78. 7 Feigl F. Z s t f a n g e w Chemie 1931, No. 36. 8 The figures given in Tables 1 and 2 represent the sum of two consecutive titrations (15 cm 3 each, i.e. a total of 30 cm3). The latter thus contain 0.3 g of urea, neutralized in form of ammonia with 100 cm 3 of n/10 HC1 (according to Mar-
schall, 0.003 mg of urea is equivalent to 1 cm 3 of n/10
HCI). 9 Sommer I, Myrbaeck K. Hoppe Seylers Z Physiol Chem 1930, 189. 10 ibid. 11 ibid. 12 Pincussen H. Mikromethodik 1930. 13 Wohlgemuth 1. Fermentmethoden 1913; see also H a t a Z. Biochem Zst 1909, 17. 14 Stassano. Comptes rend Soc biol 1905, 58. 15 Richer C. Comptes rend de l'Acad des Sciences 1892, 114. 16 Schade H. (Russian). Physical Chemistry in Medicine 1928. 17 Schulz H. 'Pharmakotherapie'. 18 Koenig K. Z s t f g e s exp Med 1927, 56. 19 Junker N. Biol Zentralblatt 1925, 45. 20 In our view there is a close connection between these results and the instructive work done by Bier et al. on the therapeutic effect of microdoses of sulphur in furunculosis, erysipelas and septic conditions; this furnishes definite proof that sulphur is highly specific for skin diseases or in other words skin diastase. (See Bier A. M M W 1930, Nos. 36 and 38; Bier A, Bumne E. Med Klin 1928 No. g; Beck S. M M W 1930 No. 39). 21 Krawkow N. Zst ges exper Med 1923, 34. 22 J Amer Chem Soc 1916, 38; 1917, 39; 1918, 40. 23 Dilutions between those shown in the graphs were not investigated. It is certainly possible that the above fluctuations recur between the given dilutions (and if the graphs were continued to show even higher dilutions). 24 Keeser E. Arch exper Pathologic u. Pharmakologie 1931, 160. Willstaetter R, Rohdewald M. Hoppe Seylers Z Physiol Chem 1931, 203. 25 Ziegler K, Doerie M. Zstges exper Med 1931, 78. 26 Waldschmidt-Leitz E, Purr A. Ber 1929, 62. Abderhalden E, Schwab E. Fermentforschung 1931, 12. 27 Canals E, Cabanes E. Bull Soc chimie bio11932, XIV, No. 2. 28 Michaelis L, Dawidson H. Biochem Ztschr 1911, 36. Oppenheimer C. Die Fermente und ihre Wirkungen, 1926. 29 H 6 d o n L. Compt rend Soc biol 1923, 88. 30 Mislowitzer E. 'Determination of hydrogen ion concentrations in fluids' (in Russian), 1932. 31 Bier A. M M W 1930 No. 38. 32 Collago I, Dobreff M. Biochem Ztschr 1925, 165. 33 Muehlbock O , Kaufmann C. Bioch Zst 1931, 238. 34 Abderhalden E, Franke K. 'Fermentforschung' 1928, 9.