- Email: [email protected]
Modification of 4 MHz NMR water proton relaxation times in highly diluted aqueous solutions
Volume 84, Number3, July 1995
o f t e c h n o l o g y , by s c i e n t i f i c p o m p and appearances, rather than by real knowledge. We should now be able to match our soul with our real learning. Then, at last, reason could play in tune. Modification of 4 MHz N M R water proton relaxation times in highly diluted aqueous solutions J.L. DEMANGEAT, P. GRIllS, B. POITEVIN (France)
Aim and Methodology NMR T1 and T2 hydrogen proton relaxation times and T1/T2 ratio are related to the organization and dynamics of water in the vicinity of solute molecules; they differ from those of bulk water and water in the pure state. In liquid water at room temperature T1 equals T2. Lowering the temperature or introducing a solute induces organization of the water molecules leading to increased proton relaxation time. Thus T2 decreases more than T1, and the T1/T2 ratio increases. NMR is one of several tools to study bound water. In the present study, NMR relaxation tectmique was applied to very high aqueous dilutions of various substances in order to study water organization. Relaxation being strongly affected by the presence of paramagnetic substances, particular attention must be paid to such contaminants in the field of high dilutions where minor modifications are expected. Dissolved atmospheric molecular oxygen is the most common origin of perturbation of relaxation times. For that reason we examined the influence of the physical factors involved in the preparation of potencies (atmospheric pressure, duration of Vortex agitation, successive centesimal dilution/agitation process) on the amount of dissolved O 2 and on T2 relaxation time. As we found a strong influence, the rigorously defined protocol required samples to be prepared within 1 day under several strictly controlled conditions (ether cleaned and autoclaved NMR Brucker PC 7.5 mm diameter tubes, 25 seconds agitation with VortexGenie at constant rate 5, discarding plastic tip after each operation, laminar-flow exhaust hood, temperature and pressure regularly noted, filtration by 0.22 gm millipore, immediate flame-sealing); in spite of these conditions, variations of about 2-2.5 % pO 2 leading to fluctuations up to 25 msec in T1 and 12 msec in T2 at I~ were to be expected. In
169 addition, samples were labelled with code numbers for NMR measurements. NMR measurements were made on a 4 MHz Brucker PC104 Minispec connected to a DRUSCH EAF 16A 0.09406 Tesla resistive electromagnet. A constant temperature of 1 + 0.2~ was achieved by circulation of cryostatic proton-free fluid frequently controlled by thennocouple. T1 was determined by computerized monoexponential fitting of an 8-point experimental curve and averaged from at least 10 to 15 successive measurements. T2 was d e t e r m i n e d by the CarrPurcell-Meiboom-Gill sequence and monoexponential fitting of a 160-point experimental curve. Each sample was measured at least 15 to 20 times. First series We studied silica/lactose (Sil/Lac) saline potencies one of us had previously shown to be active at very high dilution on mouse peritoneal macrophages when injected iutraperitoneally or orally administered in drinking water; the production of paf-acether, a mediator in allergy and inflammation, was significantly increased in the isolated, zymosanstimulated murine macrophages. The effect was found at a dilution level as high as 10-18 (9cH). The involvement of silica as an active constituent was demonstrated by controls r e c e i v i n g p h y s i o l o g i c a l salt, lactose or Gelsemium at the same level of dilution. Besides, in homoeopathic practice the effect of Silicea (Sil/Lac) in recurring infections and chronic suppuration is a regular clinical observation. Silicon dioxide (insoluble) was homogenized by 3 successive triturations with pure lactose and then added to 0.9% NaC1 (0.3 g in 3 ml). This initial solution was successively diluted 13 times by adding 0.3 ml to 30 ml of 0.9% NaC1 (Aguettaut, pH 6.0-6.9). Only 5 centesimal dilutions were retained (3cH, 6cH, 9cH, 12 cH, 15cH), containing theoretical silica concentrations of 1.66.10 .5 M to 1.66.10 -29 M and lactose concentrations of 2.92.10 3 M to 2.92.10 -27 M. As references, 0.9% NaC1 and distilled water (Meram, pH 6.5-7.5) were subjected simultaneously to identical cycles o f dilution/agitation and stored in code-labelled NMR tubes just as for Sil/Lac. All samples of a given series (Sil/Lac,
170 saline, water) were prepared on the same day to ensure a similar level of dissolved atmospheric 0 2. 7 independent series of all samples were p r e p a r e d on a monthly basis, using new batches of solvent each time. Second series In 3 new independent series of Sil/Lac, saline and water, additional m a n g a n e s e / l a c t o s e (Mn/Lac) and histamine (Hist) dilutions were prepared under the same conditions except that the solvent was water instead of saline and the first potency was 4cH instead of 3cH (to minimize a potential effect of the solute on the NMR signal). As manganese is insoluble, Mn/Lac was prepared from triturations in lactose, as for Sil/Lac. In the final retained samples, manganese concentrations ranged from 1.82"10 -7 M to 1.82"10 -29 M and lactose concentrations from 2.92"10 -6 M to 2.92"10 -28 M; histamine ranged from 9.8"10 -8 M to 9.8"10 -30 M. Results First series - - 0 . 9 % NaC1 differed from water in having a lower T2 (1365.6 :~ 30.0 ms and 1415.1 • 32.0 ms respectively; p<0.0001) and a higher T1/T2 ratio (1.127 • 0.046 and 1.082 • 0.032 respectively; p<0.0001); such a result was expected, reflecting orientation and reduction in mobility of the dipolar water molecules in the powerful electric field of Na+ and C1- ions. --Sil/Lac differed from its saline solvent by higher T1 (1570.1 • 72.7 ms and 1533.4 • 66.6 ms respectively; p<0.034) and T1/T2 ratio (1.148 • 0.053 and 1.121• 0.036 respectively; p<0.018) without significant variation in T2. Moreover, differences persisted when the least diluted samples (3cH and 6cH), possibly containing solute, were excluded from the analysis (significant levels being p<0.039 for T1 and p<0.025 for T 1 / T 2 ) , in the Sil/Lac d i l u t i o n s thus remaining under consideration and which were still different from the solvent, there only remained theoretical concentrations o f less than 1.66"10 -17 M silica and 2.92"10 -15 M lactose. - - T 1 in Sil/Lac dilutions was paradoxically higher than in pure water (1570.1 • 72.7 ms and 1526.3 • 57.0 ms respectively).
British HomoeopathicJournal
Second series - - N o significant differences in T1 were found between Sil/Lac and 0.9% NaCI probably due to an inadequate number of samples; but discriminant analysis showed separation. - - T 1 was increased (n.s.) in Mn/Lac and Hisst compared to water (1613.21 + 26.21 ms; 1619.72 • 35.04 ms respectively; water = 1575.71 • 77.5 ms) aswell as in T1/T2 ratio (1.035 • 0.03 and 1.034 • 0.04 respectively; water = 1.019 • 0.03). - - U s i n g analysis o f variance, significant g l o b a l d i f f e r e n c e s ( p < 0 . 0 2 4 for T1; p<0.037 for T1/T2) between substances curiously occur only above the 9cH. --Discriminant analysis moreover separated Mn/Lac and Hist from water (p<0.005). Conclusion T1 NMR hydrogen proton relaxation time is increased in very high dilutions of various solutes with the solvent prepared and measured in identical conditions. Differences persist at dilution levels higher than 10 -18. Many chemicophysical parameters were analysed. No explanation can be derived from our experiments at present. The results nevertheless encourage the use of NMR for further investigation of homoeopathic solutions.
Non-linear thermodynamics, quantum physics, eleetrophysiology and self-organization phenomena as a framework for looking at the organism and its pathology in a holistie context A.N. DEL1NICK (Greece) The aim of this paper is to provide a model of the organism and its pathology that can be used by h o m o e o p a t h s and researchers in order to better understand the workings of the organism through a holistic framework. It aims to explain the Arndt-Schulz Law formulated in 1888 and lately revived as 'hormesis'. The Arndt-Schulz Law, one of the earliest laws of pharmacology, states that for every substance, small doses stimulate, moderate doses inhibit and large doses kill. This has been revived recently in the field of toxicology as hormesis, the term used for the stimulatory effect low levels of usually poisonous substances have on an organism. Conventional drugs tend to be in the moderate dose r a n g e o f the A r n d t - S c h u l z c u r v e w h i l e
o f t e c h n o l o g y , by s c i e n t i f i c p o m p and appearances, rather than by real knowledge. We should now be able to match our soul with our real learning. Then, at last, reason could play in tune. Modification of 4 MHz N M R water proton relaxation times in highly diluted aqueous solutions J.L. DEMANGEAT, P. GRIllS, B. POITEVIN (France)
Aim and Methodology NMR T1 and T2 hydrogen proton relaxation times and T1/T2 ratio are related to the organization and dynamics of water in the vicinity of solute molecules; they differ from those of bulk water and water in the pure state. In liquid water at room temperature T1 equals T2. Lowering the temperature or introducing a solute induces organization of the water molecules leading to increased proton relaxation time. Thus T2 decreases more than T1, and the T1/T2 ratio increases. NMR is one of several tools to study bound water. In the present study, NMR relaxation tectmique was applied to very high aqueous dilutions of various substances in order to study water organization. Relaxation being strongly affected by the presence of paramagnetic substances, particular attention must be paid to such contaminants in the field of high dilutions where minor modifications are expected. Dissolved atmospheric molecular oxygen is the most common origin of perturbation of relaxation times. For that reason we examined the influence of the physical factors involved in the preparation of potencies (atmospheric pressure, duration of Vortex agitation, successive centesimal dilution/agitation process) on the amount of dissolved O 2 and on T2 relaxation time. As we found a strong influence, the rigorously defined protocol required samples to be prepared within 1 day under several strictly controlled conditions (ether cleaned and autoclaved NMR Brucker PC 7.5 mm diameter tubes, 25 seconds agitation with VortexGenie at constant rate 5, discarding plastic tip after each operation, laminar-flow exhaust hood, temperature and pressure regularly noted, filtration by 0.22 gm millipore, immediate flame-sealing); in spite of these conditions, variations of about 2-2.5 % pO 2 leading to fluctuations up to 25 msec in T1 and 12 msec in T2 at I~ were to be expected. In
169 addition, samples were labelled with code numbers for NMR measurements. NMR measurements were made on a 4 MHz Brucker PC104 Minispec connected to a DRUSCH EAF 16A 0.09406 Tesla resistive electromagnet. A constant temperature of 1 + 0.2~ was achieved by circulation of cryostatic proton-free fluid frequently controlled by thennocouple. T1 was determined by computerized monoexponential fitting of an 8-point experimental curve and averaged from at least 10 to 15 successive measurements. T2 was d e t e r m i n e d by the CarrPurcell-Meiboom-Gill sequence and monoexponential fitting of a 160-point experimental curve. Each sample was measured at least 15 to 20 times. First series We studied silica/lactose (Sil/Lac) saline potencies one of us had previously shown to be active at very high dilution on mouse peritoneal macrophages when injected iutraperitoneally or orally administered in drinking water; the production of paf-acether, a mediator in allergy and inflammation, was significantly increased in the isolated, zymosanstimulated murine macrophages. The effect was found at a dilution level as high as 10-18 (9cH). The involvement of silica as an active constituent was demonstrated by controls r e c e i v i n g p h y s i o l o g i c a l salt, lactose or Gelsemium at the same level of dilution. Besides, in homoeopathic practice the effect of Silicea (Sil/Lac) in recurring infections and chronic suppuration is a regular clinical observation. Silicon dioxide (insoluble) was homogenized by 3 successive triturations with pure lactose and then added to 0.9% NaC1 (0.3 g in 3 ml). This initial solution was successively diluted 13 times by adding 0.3 ml to 30 ml of 0.9% NaC1 (Aguettaut, pH 6.0-6.9). Only 5 centesimal dilutions were retained (3cH, 6cH, 9cH, 12 cH, 15cH), containing theoretical silica concentrations of 1.66.10 .5 M to 1.66.10 -29 M and lactose concentrations of 2.92.10 3 M to 2.92.10 -27 M. As references, 0.9% NaC1 and distilled water (Meram, pH 6.5-7.5) were subjected simultaneously to identical cycles o f dilution/agitation and stored in code-labelled NMR tubes just as for Sil/Lac. All samples of a given series (Sil/Lac,
170 saline, water) were prepared on the same day to ensure a similar level of dissolved atmospheric 0 2. 7 independent series of all samples were p r e p a r e d on a monthly basis, using new batches of solvent each time. Second series In 3 new independent series of Sil/Lac, saline and water, additional m a n g a n e s e / l a c t o s e (Mn/Lac) and histamine (Hist) dilutions were prepared under the same conditions except that the solvent was water instead of saline and the first potency was 4cH instead of 3cH (to minimize a potential effect of the solute on the NMR signal). As manganese is insoluble, Mn/Lac was prepared from triturations in lactose, as for Sil/Lac. In the final retained samples, manganese concentrations ranged from 1.82"10 -7 M to 1.82"10 -29 M and lactose concentrations from 2.92"10 -6 M to 2.92"10 -28 M; histamine ranged from 9.8"10 -8 M to 9.8"10 -30 M. Results First series - - 0 . 9 % NaC1 differed from water in having a lower T2 (1365.6 :~ 30.0 ms and 1415.1 • 32.0 ms respectively; p<0.0001) and a higher T1/T2 ratio (1.127 • 0.046 and 1.082 • 0.032 respectively; p<0.0001); such a result was expected, reflecting orientation and reduction in mobility of the dipolar water molecules in the powerful electric field of Na+ and C1- ions. --Sil/Lac differed from its saline solvent by higher T1 (1570.1 • 72.7 ms and 1533.4 • 66.6 ms respectively; p<0.034) and T1/T2 ratio (1.148 • 0.053 and 1.121• 0.036 respectively; p<0.018) without significant variation in T2. Moreover, differences persisted when the least diluted samples (3cH and 6cH), possibly containing solute, were excluded from the analysis (significant levels being p<0.039 for T1 and p<0.025 for T 1 / T 2 ) , in the Sil/Lac d i l u t i o n s thus remaining under consideration and which were still different from the solvent, there only remained theoretical concentrations o f less than 1.66"10 -17 M silica and 2.92"10 -15 M lactose. - - T 1 in Sil/Lac dilutions was paradoxically higher than in pure water (1570.1 • 72.7 ms and 1526.3 • 57.0 ms respectively).
British HomoeopathicJournal
Second series - - N o significant differences in T1 were found between Sil/Lac and 0.9% NaCI probably due to an inadequate number of samples; but discriminant analysis showed separation. - - T 1 was increased (n.s.) in Mn/Lac and Hisst compared to water (1613.21 + 26.21 ms; 1619.72 • 35.04 ms respectively; water = 1575.71 • 77.5 ms) aswell as in T1/T2 ratio (1.035 • 0.03 and 1.034 • 0.04 respectively; water = 1.019 • 0.03). - - U s i n g analysis o f variance, significant g l o b a l d i f f e r e n c e s ( p < 0 . 0 2 4 for T1; p<0.037 for T1/T2) between substances curiously occur only above the 9cH. --Discriminant analysis moreover separated Mn/Lac and Hist from water (p<0.005). Conclusion T1 NMR hydrogen proton relaxation time is increased in very high dilutions of various solutes with the solvent prepared and measured in identical conditions. Differences persist at dilution levels higher than 10 -18. Many chemicophysical parameters were analysed. No explanation can be derived from our experiments at present. The results nevertheless encourage the use of NMR for further investigation of homoeopathic solutions.
Non-linear thermodynamics, quantum physics, eleetrophysiology and self-organization phenomena as a framework for looking at the organism and its pathology in a holistie context A.N. DEL1NICK (Greece) The aim of this paper is to provide a model of the organism and its pathology that can be used by h o m o e o p a t h s and researchers in order to better understand the workings of the organism through a holistic framework. It aims to explain the Arndt-Schulz Law formulated in 1888 and lately revived as 'hormesis'. The Arndt-Schulz Law, one of the earliest laws of pharmacology, states that for every substance, small doses stimulate, moderate doses inhibit and large doses kill. This has been revived recently in the field of toxicology as hormesis, the term used for the stimulatory effect low levels of usually poisonous substances have on an organism. Conventional drugs tend to be in the moderate dose r a n g e o f the A r n d t - S c h u l z c u r v e w h i l e