Category Archives: Vol 5 | Issue 1 | Jan- Apr 2017

“In – Vitro Studies on Inhibition of Acetyl Cholinesterase Activities of Marine Organisms by Organophosphorus Pesticides”


Vol 5 | Issue 1 | Jan - Apr 2017 | page: 21-26 | Rumi Dasgupta


Author: Rumi Dasgupta [1]

[1] University of East London, Stratford, London.
Institute Where Research Was Conducted: School of Health, Sports and Biosciences, University of East London, Stratford, London.
University Affiliation: University of East London.
Year Of Acceptance Of Thesis: 2009.

Address of Correspondence
Dr. Rumi Dasgupta
Research Trainee, MS (Toxicology), University of East London,
Stratford, London.
Email: rumidasgupta@gmail.com


Abstract

Background: AChE activities are known to vary according to seasons with the highest values being found during summer. This is particularly true for cholinesterase activity in fish. Most enzymatic activities in poikilothermic species vary with the temperature of their environment. The level of cholinesterase activity does not directly depend on ambient temperature but on the physiological activity, which is tightly correlated with water temperature. Variations of biotic parameters such as sex, size, age, genital maturity or starvation are known to influence biological markers and make the environmental significance of markers difficult to interpret. The inhibition of AChE activities by pesticides and toxic elements like cadmium, lead and copper is well known and its use as a specific biomarker of exposure of aquatic organisms to such pollutants is widely applied in laboratory and field studies. The present work presents an evaluation of the acetyl cholinesterase activities of different species of marine organisms.
Method: Sampling was done from two Goan beaches for diversity in several physiological parameters like water quality, dissolved oxygen and contaminants disposal. Samples of Nerita chameleon and Etroplus suratensis were collected for the study. Enzymes were extracted by puncturing the forebrain region and homogenised. Protein estimation was carried out by using the Lowry Assay. The pesticides: Methyl Parathion, Ethyl Parathion, Phosphomidon, Monocrotophos and Dichlorovos were studied. Inhibition of AChE activity was determined by finding the difference in pH and % inhibition.
Results: In case of Nerita chameleon the order of toxicity is: DDVP > Ethyl Parathion > Phosphoamidon ~ Methyl Parathion whereas in case of Etroplus suratensis the order of toxicity is: Methyl Parathion > Monocrotophos > Phosphoamidon > Ethyl Parathion > DDVP. Out of the two samples it is found that the level of toxicity varies from species to species but the pesticides have an adverse effect on the activity of AChE leading to neurological disorders like Myasthenia gravis, glaucoma & Alzheimer's disease, etc.
Conclusion: Current sets of observations show that the study of Acetylcholinesterase enzyme can help the Eco-toxicologists to assess the impacts of various lethal & sub-lethal pollution in the marine environment. Study of different marine organisms like molluscs, fish, echinoderms, etc. can help to assess the biological effects of the neurotoxic contaminants on marine & estuarine environment.
Keywords: AChE, marine pollution, Delta pH-metric method.

Thesis Question: 1. To determine activity of acetylcholinesterase activity.
2. To evaluate the inhibitory effects of various neurotoxic contaminants on acetylcholinesterase activity.
3. To assess impact of neurotoxic contaminants on marine fishes along the Goa coast in terms of acetylcholinesterase activities
Thesis Answer: Current sets of observations show that the study of Acetylcholinesterase enzyme can help the Eco-toxicologists to assess the impacts of various lethal & sub-lethal pollution in the marine environment. Study of different marine organisms like molluscs, fish, echinoderms, etc. can help to assess the biological effects of the neurotoxic contaminants on marine & estuarine environment.


THESIS SUMMARY

Introduction

Introduction of man, directly or indirectly, of substances or energy into the marine environment resulting in toxic effects harmful to living resources, hazard to human health, hindrance to marine activities like fishing, loss of quality for use of sea-water and reduction of amenities. The marine environment is under continuous stress of various types of contaminants due to discharge of waste materials, industrial effluents, municipal sewage, riverine runoff, agricultural runoff etc. The various types of contaminants that are generally found in the marine environment include organic such as organochlorine pesticides, organophosphorus pesticides, polychlorinated biphenyls, polychlorinated dibenzo dioxin, dibenzo furans, polycyclic aromatic hydrocarbon, and inorganic contaminants like lead, cadmium, mercury, copper, etc. which are highly toxic.
The water is most polluted in the coastal sides but as the water deepens the pollutants or chemicals gets diluted. Since the water is most polluted in the coastal side, thus, it has an adverse effect on organisms like fishes, oysters, etc. We take in those fishes which already are contaminated with deposition of metals or other toxicants which results in various abnormalities. To detect the presence of toxic pollutants various methods are there out of which biomarker is one of them. It can be done by degrading bacteria, or by using various enzymes or by means of chemicals.
Biomarker is specific in nature. Each biomarker can detect only a particular abnormality. For e.g. AChE. Acetyl cholinesterase or AChE hydrolyses Acetyl Choline during motor impulses in our body. Now if some neurotoxic contaminants are present then due to the blockage of active sites the activity of AChE gets reduced leading to neurological disorders. Thus, AChE acts as a biomarker for neurotoxic contaminants like organophosphorus pesticides, heavy metals like Pb, Cd, etc.
For decades, the measurement of ChE is a valuable tool that should be incorporated to a battery of biomarkers to maximize the confidence with which eco-toxicologists assess impacts of sub-lethal pollution in the marine and estuarine environment. As used in other organisms, like molluscs and echinoderms, and fish[1], AChE appears as a relevant means of investigating biological effects of many neurotoxic contaminants on aquatic habitats and tropic levels[2].
Quantifying brain or plasma Cholinesterase (ChE) enzyme activity is widely used method to monitor avian exposure to OP pesticides[3][4]. Enzyme cholinesterase activity related to pesticides effects has been measured in brain [5][6][7], gills [6], liver [7], heart [8], muscle [7][8][9] and erythrocyte of different fresh water species.
Studies by Sengupta R. et al. comparing the sensitivity of different species towards several pesticides revealed that invertebrate ChEs, namely insect AChE, were more sensitive to inhibition than those from vertebrates[10]. Therefore, prior to the use of an invertebrate ChE as a reliable biomarker, several features like tissue distribution, enzymatic behaviour, and optimal conditions for assessing activity and sensitivity to anti-cholinesterase pesticides should be investigated[11][12][13].

Aims & Objectives

In view of the ongoing problem of marine pollution due to neurotoxic contaminants it is indeed a prime need of the hour to assess the impact of such contamination by toxic compounds using various biomarker techniques. To evaluate the toxicological impact of various types of contaminants, I have been prompted to undertake a systematic study on biomarker of pollution. The main aims and objective the present studies are as follows:
4. To determine activity of acetylcholinesterase activity by Delta pH-metric Method
5. To evaluate the inhibitory effects of various neurotoxic contaminants on acetylcholinesterase activity.
6. To assess impact of neurotoxic contaminants on marine fishes along the Goa coast in terms of acetylcholinesterase activities.

Materials and Methods

1. Sampling Areas:
Sampling was done from 2 beaches of Goan Coastal area. First one is the local site Dona Paula Beach few 2 kilometres away from NIO. The second sampling area is Madkai. Thus, both the sites were far enough to show diversity in several physiological parameters such as water quality, dissolved oxygen, and disposal of contaminants.
2. Animal Samples:
a. Nerite chameleon
b. Etroplus suratensis
3. Extraction of Enzymes:
a. As soon as the organisms are collected, they are subjected to about 4°C and rest of the process is carried in the same set of conditions. The scales are removed from the surface of the fishes and the abdominal muscles are taken out. Similarly, the skull is punctured at the forebrain region and slowly the upper skull is removed and the whole brain along with the Pons is collected.
b. As soon as the snails are collected, their weight and dimensions are measured along with their shells. Then their shells are broken & tissues are weighed.
c. To each homogenate tube, about 4 g of tissue is added along with 4ml of extraction buffer (pH-7.4), 4 ml of Sucrose and a drop of Triton X-100. Each tube is subjected to homogenization (Homoginizer-Ultra Durax), at sheer speed of 11000 per minute, thrice for one minute with an interval of 30 seconds. The Homogenization is followed by cold centrifugation at -4°c for 30 minutes at a speed of 18500rpm. After the process of homogenization, the supernatant is pipetted out and dispensed in cryovoils and stored at -20°C.
4. Estimation of Protein Content of the Extract by Hartree-Lowry Assay:
The Lowry assay (1951) is used to estimate protein content of the enzyme extract. It is the method of choice for accurate protein estimation for cell fractions[14]. The Hartree version of the Lowry assay uses fewer reagents, improves sensitivity and provides a more linear response.
5. Experimental Setup:
The reaction was carried out in small glass aliquots. To each aliquot < 7ml of Working Buffer (pH-8.02) was added followed 0.100 ml of enzyme and inhibitors if required were added. Considering the volume of the substrate to be added later the final is made up to 7.5ml.
Finally, the reaction was commenced by addition of the substrate. First reading is taken as soon as the substrate is added, and readings were taken after every subsequent 10 minutes’ interval.
The colour, which is initially dark blue gradually, changes to green and finally to golden yellow as the reaction reaches towards completion. Since the working buffer contains an indicator Bromothymol blue, which imparts a blue colour to a basic solution and gradually change its spectrum to yellow as the solution becomes acidic.
6. Organosphosphate Pesticides:
a. Methyl Parathion
b. Ethyl Parathion
c. Phosphomidon
d. Monocrotophos
e. Dichlorovos
7. Determination of Acetylcholinesterase Activity by Delta pH-metric Method:
The change in the pH is due to the liberation of Acetic acid. The release of acetic acid is due to the hydrolysis of the substrate Acetylcholine Bromide or Butyl-Choline-thio-Iodide, by the enzyme present in the tissue homogenate in the presence of NaCl. Acetylcholinesterase activity was measured following modified delta-pH metric method[15] in which 0.1 ml of sample enzyme was incubated at room temperature (30◦C) with 0.2 ml substrate acetylcholine bromide for an hour in a medium of phosphate buffer (0.01 M, pH-8.0 ± 0.10) indicator solution with the final volume being 7.5 ml. Bromothymol blue was used as the indicator for observation of the reaction kinetics. The changes in pH were recorded at an interval of 10 minutes. The difference in pH changes over a period of one hour corresponded to the amount of acetic acid liberated due to reaction kinetics with the acetylcholinesterase enzyme. The unit of activity of acetylcholinesterase was calibrated with respect to micromoles of acetic acid liberated by the action AChE enzyme. The protein concentration of each of the sample extract was determined as per Lowry et. al. using Bovine serum albumin as the standard[14]. The AChE activities are expressed in terms of the units per mg of protein per minute.
The amount of the acetic acid released per unit time is directly proportional to the number of substrate molecules hydrolysed (turnover number) and this release taken as the ΔpH at the end of the reaction after one hour per mg of protein is the actual measure of the activity of enzyme.
Enzyme activity (Units/mg - protein/hour)
= [(56.246 × ΔpH) × y / (Protein content × 60)] × Working Volume
Where y = Volume of the enzyme used.
The 50% inhibition concentration (IC50) and double reciprocal plots were determined for these compounds. The aim of the present study is to determine the relative toxicity of neurotoxic metals, pesticides and OP compounds as related to the comparative neurotoxic potential and the mechanism of AChE potential in different tissues of the fishes.
The aspects show that the measurement of ‘total cholinesterase activity’ under acute assay conditions in homogenates prepared with whole animals, can lead to erroneous ecotoxicological conclusions[9].

Results

To eliminate the factor concerning the effect of solvent on inhibition of AChE activity, I have taken 50ul of acetone + 100µl of Enzyme + 200µlof Substrate + 150µl of Distilled water (i.e. Control 2). The result shows that there is no change in AChE activity due to the presence of Acetone in the Enzyme-Substrate buffer medium.
Measurement of AChE activity was done by measuring the pH reading every 10 minutes.
1. Inhibition of AChE activity of Nerita chamaeleon by Phosphoamidon in a concentration of 1x10-6M collected from location: Dona Paula. The date of collection of the sample was 17.06.2006.
a. The Blank pH started with a pH reading of 8.08 at 0 mins and at 60 mins the reading was 8.07. The delta pH was 0.01.
b. The Blank + Enzyme pH started with a pH reading of 7.86 at 0 mins and at 60 mins the reading was 7.59. The delta pH was 0.27.
c. The Blank + Substrate pH started with a pH reading of 7.96 at 0 mins and at 60 mins the reading was 6.46. The delta pH was 1.50.
d. The Control pH started with a pH reading of 7.18 at 0 mins and at 60 mins the reading was 5.62. The delta pH was 1.56.
e. The Sample 1 pH started with a pH reading of 7.09 at 0 mins and at 60 mins the reading was 5.71. The delta pH was 1.38 and the % Inhibition was found to be 11.53%.
f. The Sample 2 pH started with a pH reading of 7.11 at 0 mins and at 60 mins the reading was 5.78. The delta pH was 1.33 and the % Inhibition was found to be 14.74%.
g. The Sample 3 pH started with a pH reading of 7.03 at 0 mins and at 60 mins the reading was 5.73. The delta pH was 1.30 and the % Inhibition was found to be 16.67%.
h. The Sample 4 pH started with a pH reading of 7.11 at 0 mins and at 60 mins the reading was 5.85. The delta pH was 1.26 and the % Inhibition was found to be 19.23%.
i. The Sample 5 pH started with a pH reading of 7.15 at 0 mins and at 60 mins the reading was 6.05. The delta pH was 0.59 and the % Inhibition was found to be 62.17%.

2. Inhibition of AChE activity of Nerita chamaeleon by Methyl Parathion in a concentration of 1x10-8M collected from location: Dona Paula. The date of collection of the sample was 17.06.2006.
a. The Blank pH started with a pH reading of 8.02 at 0 mins and at 60 mins the reading was 8.00. The delta pH was 0.02.
b. The Blank + Enzyme pH started with a pH reading of 7.29 at 0 mins and at 60 mins the reading was 7.09. The delta pH was 0.20.
c. The Blank + Substrate pH started with a pH reading of 7.36 at 0 mins and at 60 mins the reading was 6.46. The delta pH was 1.50.
d. The Control pH started with a pH reading of 6.72 at 0 mins and at 60 mins the reading was 6.10. The delta pH was 0.62.
e. The Sample 1 pH started with a pH reading of 6.92 at 0 mins and at 60 mins the reading was 6.32. The delta pH was 0.60 and the % Inhibition was found to be 3.22%.
f. The Sample 2 pH started with a pH reading of 6.85 at 0 mins and at 60 mins the reading was 6.26. The delta pH was 0.59 and the % Inhibition was found to be 4.83%.
g. The Sample 3 pH started with a pH reading of 6.79 at 0 mins and at 60 mins the reading was 6.23. The delta pH was 0.56 and the % Inhibition was found to be 9.67%.
h. The Sample 4 pH started with a pH reading of 6.86 at 0 mins and at 60 mins the reading was 6.32. The delta pH was 0.54 and the % Inhibition was found to be 12.9%.
i. The Sample 5 pH started with a pH reading of 6.74 at 0 mins and at 60 mins the reading was 6.32. The delta pH was 0.42 and the % Inhibition was found to be 32.25%.

3. Inhibition of AChE activity of Etroplus suratensis by DDVP in a concentration of 1x10-6M collected from location: Madkai. The date of collection of the sample was 08.06.2006.
a. The Blank pH started with a pH reading of 7.64 at 0 mins and at 60 mins the reading was 7.63. The delta pH was 0.01.
b. The Blank + Enzyme pH started with a pH reading of 6.43 at 0 mins and at 60 mins the reading was 6.36. The delta pH was 0.07.
c. The Blank + Substrate pH started with a pH reading of 7.60 at 0 mins and at 60 mins the reading was 7.15. The delta pH was 0.45.
d. The Control pH started with a pH reading of 6.35 at 0 mins and at 60 mins the reading was 5.30. The delta pH was 1.05.
e. The Sample 1 pH started with a pH reading of 6.36 at 0 mins and at 60 mins the reading was 5.43. The delta pH was 0.93 and the % Inhibition was found to be 11.42%.
f. The Sample 2 pH started with a pH reading of 6.38 at 0 mins and at 60 mins the reading was 5.47. The delta pH was 0.91 and the % Inhibition was found to be 13.33%.
g. The Sample 3 pH started with a pH reading of 6.39 at 0 mins and at 60 mins the reading was 5.50. The delta pH was 0.89 and the % Inhibition was found to be 15.23%.
h. The Sample 4 pH started with a pH reading of 6.44 at 0 mins and at 60 mins the reading was 5.82. The delta pH was 0.62 and the % Inhibition was found to be 40.95%.

4. Inhibition of AChE activity of Etroplus suratensis by Ethyl Parathion in a concentration of 1x10-6M collected from location: Madkai. The date of collection of the sample was 08.06.2006.
a. The Blank pH started with a pH reading of 7.91 at 0 mins and at 60 mins the reading was 7.87. The delta pH was 0.04.
b. The Blank + Enzyme pH started with a pH reading of 6.81 at 0 mins and at 60 mins the reading was 6.53. The delta pH was 0.28.
c. The Blank + Substrate pH started with a pH reading of 7.71 at 0 mins and at 60 mins the reading was 7.22. The delta pH was 0.49.
d. The Control pH started with a pH reading of 6.58 at 0 mins and at 60 mins the reading was 5.29. The delta pH was 1.39.
e. The Sample 1 pH started with a pH reading of 6.60 at 0 mins and at 60 mins the reading was 5.63. The delta pH was 0.97 and the % Inhibition was found to be 30.21%.
f. The Sample 2 pH started with a pH reading of 6.61 at 0 mins and at 60 mins the reading was 5.71. The delta pH was 0.90 and the % Inhibition was found to be 35.25%.
g. The Sample 3 pH started with a pH reading of 6.63 at 0 mins and at 60 mins the reading was 5.84. The delta pH was 0.79 and the % Inhibition was found to be 43.16%.
h. The Sample 4 pH started with a pH reading of 6.64 at 0 mins and at 60 mins the reading was 5.88. The delta pH was 0.76 and the % Inhibition was found to be 45.32%.
i. The Sample 5 pH started with a pH reading of 6.66 at 0 mins and at 60 mins the reading was 6.11. The delta pH was 0.55 and the % Inhibition was found to be 60.43%.

5. Inhibition of AChE activity of Etroplus suratensis by Phosphoamidon in a concentration of 1x10-6M collected from location: Madkai. The date of collection of the sample was 08.06.2006.
a. The Blank pH started with a pH reading of 7.57 at 0 mins and at 60 mins the reading was 7.54. The delta pH was 0.03.
b. The Blank + Enzyme pH started with a pH reading of 6.21 at 0 mins and at 60 mins the reading was 6.09. The delta pH was 0.12.
c. The Blank + Substrate pH started with a pH reading of 7.39 at 0 mins and at 60 mins the reading was 7.18. The delta pH was 0.21.
d. The Control pH started with a pH reading of 6.07 at 0 mins and at 60 mins the reading was 4.61. The delta pH was 1.46.
e. The Sample 1 pH started with a pH reading of 6.09 at 0 mins and at 60 mins the reading was 4.82. The delta pH was 1.27 and the % Inhibition was found to be 13.01%.
f. The Sample 2 pH started with a pH reading of 6.12 at 0 mins and at 60 mins the reading was 5.07. The delta pH was 1.05 and the % Inhibition was found to be 28.08%.
g. The Sample 3 pH started with a pH reading of 6.15 at 0 mins and at 60 mins the reading was 5.16. The delta pH was 0.99 and the % Inhibition was found to be 32.19%.
h. The Sample 4 pH started with a pH reading of 6.17 at 0 mins and at 60 mins the reading was 5.38. The delta pH was 0.79 and the % Inhibition was found to be 45.89%.
i. The Sample 5 pH started with a pH reading of 6.20 at 0 mins and at 60 mins the reading was 5.71. The delta pH was 0.49 and the % Inhibition was found to be 66.43%.

6. Inhibition of AChE activity of Etroplus suratensis by Methyl Parathion in a concentration of 1x10-6M collected from location: Madkai. The date of collection of the sample was 08.06.2006.
a. The Blank pH started with a pH reading of 7.62 at 0 mins and at 60 mins the reading was 7.60. The delta pH was 0.02.
b. The Blank + Enzyme pH started with a pH reading of 6.19 at 0 mins and at 60 mins the reading was 6.14. The delta pH was 0.05.
c. The Blank + Substrate pH started with a pH reading of 7.49 at 0 mins and at 60 mins the reading was 7.17. The delta pH was 0.32.
d. The Control pH started with a pH reading of 6.01 at 0 mins and at 60 mins the reading was 4.89. The delta pH was 1.12.
e. The Sample 1 pH started with a pH reading of 6.09 at 0 mins and at 60 mins the reading was 5.11. The delta pH was 0.98 and the % Inhibition was found to be 12.5%.
f. The Sample 2 pH started with a pH reading of 6.11 at 0 mins and at 60 mins the reading was 5.31. The delta pH was 0.80 and the % Inhibition was found to be 28.57%.
g. The Sample 3 pH started with a pH reading of 6.12 at 0 mins and at 60 mins the reading was 5.58. The delta pH was 0.54 and the % Inhibition was found to be 51.78%.
h. The Sample 4 pH started with a pH reading of 6.15 at 0 mins and at 60 mins the reading was 5.79. The delta pH was 0.36 and the % Inhibition was found to be 67.85%.

7. Inhibition of AChE activity of Etroplus suratensis by Monocrotophos in a concentration of 1x10-6M collected from location: Madkai. The date of collection of the sample was 08.06.2006.
a. The Blank pH started with a pH reading of 7.46 at 0 mins and at 60 mins the reading was 7.42. The delta pH was 0.04.
b. The Blank + Enzyme pH started with a pH reading of 6.09 at 0 mins and at 60 mins the reading was 6.05. The delta pH was 0.04.
c. The Blank + Substrate pH started with a pH reading of 7.48 at 0 mins and at 60 mins the reading was 7.40. The delta pH was 0.08.
d. The Control pH started with a pH reading of 6.06 at 0 mins and at 60 mins the reading was 4.31. The delta pH was 1.75.
e. The Sample 1 pH started with a pH reading of 6.19 at 0 mins and at 60 mins the reading was 5.11. The delta pH was 1.08 and the % Inhibition was found to be 38.28%.
f. The Sample 2 pH started with a pH reading of 6.22 at 0 mins and at 60 mins the reading was 5.19. The delta pH was 1.03 and the % Inhibition was found to be 41.14%.
g. The Sample 3 pH started with a pH reading of 6.26 at 0 mins and at 60 mins the reading was 5.39. The delta pH was 0.87 and the % Inhibition was found to be 50.28%.
h. The Sample 4 pH started with a pH reading of 6.33 at 0 mins and at 60 mins the reading was 5.59. The delta pH was 0.74 and the % Inhibition was found to be 57.71%.

8. Inhibition of AChE activity of Etroplus suratensis by combination of Ethyl Parathion + Methyl Parathion in (1:1) ratio, i.e. 1ml of Ethyl Parathion & 1ml of Methyl Parathion in a concentration of 1x10-12M collected from location: Madkai. The date of collection of the sample was 08.06.2006.
a. The Blank pH started with a pH reading of 7.67 at 0 mins and at 60 mins the reading was 7.61. The delta pH was 0.06.
b. The Blank + Enzyme pH started with a pH reading of 6.06 at 0 mins and at 60 mins the reading was 6.05. The delta pH was 0.01.
c. The Blank + Substrate pH started with a pH reading of 7.47 at 0 mins and at 60 mins the reading was 7.43. The delta pH was 0.04.
d. The Control pH started with a pH reading of 5.98 at 0 mins and at 60 mins the reading was 4.41. The delta pH was 1.57.
e. The Sample 1 pH started with a pH reading of 6.08 at 0 mins and at 60 mins the reading was 5.21. The delta pH was 0.87 and the % Inhibition was found to be 44.58%.
f. The Sample 2 pH started with a pH reading of 6.02 at 0 mins and at 60 mins the reading was 5.22. The delta pH was 0.80 and the % Inhibition was found to be 49.04%.
g. The Sample 3 pH started with a pH reading of 6.09 at 0 mins and at 60 mins the reading was 5.38. The delta pH was 0.71 and the % Inhibition was found to be 54.77%.
h. The Sample 4 pH started with a pH reading of 6.18 at 0 mins and at 60 mins the reading was 5.51. The delta pH was 0.67 and the % Inhibition was found to be 57.32%.

Observations

Acetylcholine is a cholinergic neurotransmitter. It is predominant at the neuromuscular junction. Now it is seen that the activity of Acetylcholinesterase is inhibited by several neurotoxic contaminants like heavy metals like Cd, Pb, Hg, etc., organophosphorous pesticides, etc.
In in-vitro conditions experiments were performed to see the effects of different neurotoxic contaminants on marine organism, i.e., a snail (Nerita chamaeleon) and a fish (Etroplus suratensis). Result suggests that DDVP is highly toxic followed by Ethyl Parathion in case of Nerita chamaeleon. In comparison to DDVP & Ethyl Parathion, Phosphoamidon and Methyl Parathion is less toxic. The low toxicity of Phosphoamidon is due to the presence of phenyl group. The phenyl group adds to the stability of the compound.
In case of Etroplus suratensis, Methyl Parathion is the most toxic followed by Monocrotophos, Phosphoamidon and Ethyl Parathion. Here it is seen that DDVP is the least toxic as compared to Nerita chamaeleon. The toxicity of Methyl parathion in this case is about 0.15 whereas in case of Nerita chamaeleon the toxicity of Methyl parathion is about 0.032.
Another set of observations were taken to see if there is any synergistic effect of the pesticides takes place in case of Etroplus suratensis and was found that the level of toxicity goes high when two pesticides are mixed together. Compared to the combination of Ethyl parathion & Methyl parathion, the combination of DDVP & Phosphoamidon is higher.

Conclusion

1. In case of Nerita chamaeleon the order of toxicity is
• DDVP > Ethyl Parathion > Phosphoamidon ~ Methyl Parathion
2. In order of toxicity in case of Etroplus suratensis is
• Methyl Parathion > Monocrotophos > Phosphoamidon > Ethyl Parathion > DDVP
3. Out of the two samples it is found that the level of toxicity varies from species to species but the pesticides have an adverse effect on the activity of AChE leading to neurological disorders like Myasthenia gravis, glaucoma & Alzheimer’s disease, etc.
Current sets of observations show that the study of Acetylcholinesterase enzyme can help the Eco-toxicologists to assess the impacts of various lethal & sub-lethal pollution in the marine environment. Study of different marine organisms like molluscs, fish, echinoderms, etc. can help to assess the biological effects of the neurotoxic contaminants on marine & estuarine environment.

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12. Diamantino, T., C., Almeida, E., Soares, A. M. V. M., Guilhermino L., 2003. Characterization of Cholinesterases from Daphnia magna Straus and Their Inhibition by Zinc. Bull. Environ. Contam. Toxicol.71:219–225.
13. Lehtonen, K., K., Leinio¨, S., 2003. Effects of Exposure to Copper and Malathion on Metallothionein Levels and Acetylcholinesterase Activity of the Mussel Mytilus edulis and the Clam Macoma balthica from the Northern Baltic Sea. Bull. Environ. Contam. Toxicol.71:489–496.
14. Lowry O H, Rosebrough N J, Farr AL, Randall RJ. (1951) Protein measurement with the Folin phenol reagent. J Biol Chem; 193:265-275.
15. Sarkar, A. (1992) Evaluation of the toxicity of organic matter in marine sediments. Water Science and Technology. 25: 3, 255 257.

How to Cite this Article: Dasgupta R. “In – Vitro Studies on Inhibition of Acetyl Cholinesterase Activities of marine organisms by Organophosphorus Pesticides”. Journal Medical Thesis 2017 Jan-Apr ; 5(1):21-26.

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Guest Editorial: Research Hypothesis or Statistical Hypothesis?

Vol 5 | Issue 1 | Jan - Apr 2017 | page:3-6 | Dr. Rumi Dasgupta


Author: Dr. Rumi Dasgupta

Dr. Rumi Dasgupta

International Manager, Advisory Board, International Association of Scientists and Researchers
Email: rumidasgupta@gmail.com


When I started preparing for my dissertation, I got scared when I started reading up about research hypothesis and got totally jumbled up with terms like research hypothesis, null hypothesis, alternative hypothesis and statistical hypothesis. The symbols H0, HA, H1 or H2 made me more confused. The books I was referring to or the websites I have looked up to did not help me much. Things started getting clear after my research methodology lectures and when I started working on my research proposal.
There are two types of hypotheses in a research setup – Research Hypothesis and Statistical Hypothesis. When I am talking about research hypothesis, the first important factor is the research question. The research process begins with the interest in a topic or area but to define an appropriate research question knowledge and understanding of the subject plays a crucial role. As suggested by Haynes it is important to know the boundary between the current knowledge and ignorance. The challenge lies in the determination of clinical uncertainties (in a clinical study setup) to be studied and to justify the necessity for investigation while developing a research question.
It is, hence, extremely important to increase the knowledge in the field of interest. It could be done by a thorough literature review, in-depth interviews with subjects and experts in the field, being aware of the latest trends and technological advances made in the interest area. It is imperative to comprehend what has been studied about the interest area till date to further the facts that has been formerly collected on the topic. This will help formulate the research question.
The primary research question should be driven by the hypothesis rather than the data. This means the research question and the research hypothesis should be developed before the starting the study. A research hypothesis is a knowledgeable declaration that is tentatively advanced to account for realities. It is a testable question on the area of interest. If a study is used to examine the research question, then the hypothesis is known as the experimental hypothesis. The research or clinical hypothesis is developed from the research question and then the main essentials of the study — sampling method, intervention (if applicable), comparison and outcome variables — are abridged in a form that creates the basis for testing, statistical and eventually medical significance.
However, while testing the statistical significance, the theory should be stated as a “null” hypothesis. The hypothesis testing is done to infer about the study population based on random sampling. Statistical hypothesis is the statement about whether a pattern or trend or difference is present in the collected data.
The research design will help to determine the choice of statistical test to be used – a simple study design may require a student t-test while a more complex design may require ANOVA or a correlational study design may use a correlation coefficient. Here, each of these statistical tests will have a different null hypothesis and an alternative hypothesis. Statistical tests are used to differentiate between the null hypothesis and alternative hypothesis.
In an article, C. Alan Boneau stated that from the given endless piles of numbers from a small sample, the t-test and F-test will determine if the means of the piles are different or not. So, rejection of null hypothesis or the alternative hypothesis does not necessarily mean it supports the tested research hypothesis. For example, a psychiatrist may predict an interaction of the variables and may reject the null hypothesis for the interaction in ANOVA; but the alternative hypothesis for interaction in an ANOVA may simply denote an occurrence of the interaction. There could be many ways for an interaction and the observed interaction may not be the one predicted in the research hypothesis.
Moreover, it is not important for the statistical test results to address the research hypothesis. While the statistical hypothesis should be discussed with the results, research hypothesis should be discussed in the discussion and conclusion section.


How to Cite this Article: Dasgupta R. Research Hypothesis or Statistical Hypothesis?. Journal Medical Thesis 2017 Jan-Apr ; 5(1): 2.

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“Effect of early mobilization in the conservative management of stable two part fracture of surgical neck of humerus”


Vol 5 | Issue 1 | Jan - Apr 2017 | page: 18-20 | Jangle Rajendra Dnyandeo


Author: Jangle Rajendra Dnyandeo [1]

[1] Medical Officer (Orthopedics), D.N.B. (Ortho.), Mumbai Port Trust Hospital, Wadala (East), Mumbai-37.
Institute Where Research Was Conducted: Little Flower Hospital and Research Centre, Angamali, Kerala-683572
University Affiliation: National Board Of Examination, New Delhi.
Year Of Acceptance Of Thesis: 2014.

Address of Correspondence
Dr. Jangle Rajendra Dnyandeo
Medical Officer (Orthopedics), D.N.B. (Ortho.), Mumbai Port Trust Hospital, Wadala (East), Mumbai-37.
Email: rajendrajangle@gmail.com


Abstract

Background: This study was done to evaluate the effect immediate mobilization on pain, function and range of motion and to study the effect of displacement, angulation and impaction of the fracture on clinical outcome after conservative treatment.
Method: Prospective study of 26 patients of stable two part fracture of surgical neck of the humerus treated conservatively, randomized in group A i.e. patients receiving early mobilization at 1 week and group B i.e. patients receiving conventional treatment of 3 weeks immobilization followed by physiotherapy. The Constant- Murley shoulder score used for assessment and Patients were followed at 6 weeks and 12 weeks, 24 wks.
Results: Majority of patients i.e. 10 (38.5%) were from 71-80 years age group with 22 females 84.60% and 4 were males 15.40%. Constant shoulder score was better at 6 months with 78 mean for group A and 70 for group B.
Conclusion: Constant shoulder score was better at 6 months in two groups and had significant difference at 6 wks and 12 wks. Majority of the patients had angulated two part fractures of surgical neck 50% . Separated group of fracture configuration patients had statistically significant result at 24 weeks.
Keywords: Proximal humerus fractures, Conservative treatment.


THESIS SUMMARY

Introduction

Proximal humerus is the segment of the humerus obtained by erecting the square which accommodates the widest part of the proximal humerus. Fractures of the proximal humerus are common and debilitating injuries and are an increasing problem in the elderly. These fractures are best treated nonoperatively but the ideal duration of immobilization in a sling before physiotherapy begins has not been clearly defined.
Proximal humeral fractures are one of the most common osteoporotic fractures and account for 4-5% of all fractures. Approximately 40% of these fractures are displaced fractures involving surgical neck.
Literature shows that there are studies describing rehabilitation of minimally displaced fractures. But very little has been reviewed regarding rehabilitation of stable two part fracture of humerus through surgical neck treated conservatively which is quite common in practice.
So there is a need to establish a definite protocol for rehabilitation of two-part fracture of surgical neck of humerus treated non-operatively.

Aims and Objective

To study the clinical outcome after the stable fracture of surgical neck of humerus treated conservatively
Objectives-
1. To study the effect immediate mobilization on pain, function and range of motion.
2. To study the effect of displacement, angulation and impaction of the fracture on clinical outcome after conservative treatment

Materials and Methods

Study area was Department of Orthopaedics, L.F. Hospital, Angamaly, Kerala.
Study population- All patients with stable two part fracture of proximal humerus with age > 50 yrs, presenting to department of orthopaedics, Little Flower Hospital, Angamaly from august 2009 to August 2012.
Total twenty six patients were included in the study. They were included in study after applying inclusion and exclusion criteria which are as follows:

Inclusion criterion
All two part fracture of surgical neck of humerus (according to Neer’s classification) which are stable and in an acceptable position by two conditions- i) displacement (<66%) and angulation are acceptable may or may not be reduced manually and ii) the humeral head and shaft move as a unit.
Age > 50 yrs

Exclusion criterion
Patients with previous ipsilateral or contralateral shoulder problems.
Patients with ipsilateral elbow problem .
Patients with poor general health.
Pathological fractures.
Multifragmentary displaced fractures of surgical neck.
Patients unable to comprehend/ follow treatment or mobilization protocol
Baseline assessment included determination of the patient’s age, gender, dominant side, co-morbid conditions and associated injuries and the cause of the injury.

Non operative treatment protocol
Group A- patients receiving early physiotherapy by 1 week
Shoulder immobiliser for 1 week.
Phase I exercises upto 3rd week.
4th week to 3 months phase II exercises.
After 3 months phase III exercises.

Group B- immobilisation for 3weeks followed by physiotherapy
Shoulder immobiliser for 3 weeks.
4th to 6th week phase I exercises.
7th to 12th week phase II exercises,
after 3 months phase III exercises
The Constant- Murley shoulder score used for assessment. Qualitative variables were recorded as percentages, and comparisons between treatments involved the use of a chi square. Quantitative variables were recorded as the mean and the standard deviation; means were compared with use of the Independent samples t test, and mean differences were calculated with 95% confidence intervals.

Results

Age wise distribution of cases
Majority of patients i.e. 10 (38.5%) were from 71-80 years age group, followed by 7 (26.9%) patients in 81-90 years of age groups. The mean age in our study was 72.34 years. We did not found statistically significant difference in terms of Constant shoulder score outcomes amongst different age groups.

Sex wise distribution of cases
There were 22 females 84.60% and 4 were males 15.40%. We have analysed functional outcomes of males and females with help of independent T test. We found no statistically significant difference amongst two groups.
Fracture configuration-
50% were angulated fractures.We have analysed functional outcomes of the three fracture configurations with help of Chi square test. We found no statistically significant difference amongst the three groups at 6, 12 weeks but at 24 weeks separated fracture configuration group had better result compared to other two groups.
Co-morbid conditions-
One patient had dementia, two patients were had alcohol abuse while one patient developed lt lateral tibial plateau fracture at 2wks during rehabilitation period.
Outcome measure-
Constant shoulder score was better at 6 months with 78 mean for group A and 70 for group B. We have analysed outcomes of two groups with help of independent T test. We found that there is statistically significant difference amongst two groups at 6 weeks and 12 weeks with mean constant shoulder score was higher in group A that is early mobilisation group.
Complications-
All four patients with co morbid conditions had shoulder stiffness with Constant shoulder score <60 at 6 months.

Discussion

Young TB et al, Clifford PC, Kristiansen B et al have shown that there is no consensus regarding the ideal duration of immobilization in a sling before physiotherapy begins in non operatively treated two part surgical neck of humerus fractures. Present study shows that early passive shoulder joint mobilization after nonoperatively treated stable two part fractures of surgical neck of humerus offers a better chance for a more rapid gain in shoulder functional status than does classic three-week immobilization followed by physiotherapy.
In our study 50% patients had angulated fractures, 26.90% had separated fractures and 23.10% comminuted fractures. All fractures healed by 6 weeks and we did not observed any displacement. This is correlated with other studies, CourtBrown CM, Hodgson SA et al, Correard RP. When three fracture configurations were analysed in respect to Constant shoulder scores with > 60 good or excellent and < 60 as poor result, we found that at 6, 12 weeks there was no significant difference but at 24 weeks, separated group of fractures had all cases with the score above 60.
We compared the constant shoulder score at 6, 12, 24 weeks with respect to age, gender, side of involvement but there was no significant difference in Constant shoulder score as >60 or < 60. Our results suggest that immediate passive mobilization is safe for these fractures because we observed the better constant shoulder score at 6 weeks and 12 weeks. But the outcome is not significant at 24 weeks. This observation is similar to M.M. Lefevre-Colau et al. series.
In our study four patients had shoulder stiffness with constant shoulder score <60 which we considered as poor result. All four patients had relevant co-morbid conditions. One patient had dementia, two patients had alcohol abuse and one patient developed lower extremity fracture at 2weeks during rehabilitation period. In other studies co-morbid conditions have not been considered.

This study has some limitations. This study did not recorded generic health-related quality-of-life measures such as SF36 score during the follow-up period.
Another limitation was that physical therapy for both treatment groups was not uniformly supervised or unsupervised. Some patients with excellent results had received completely supervised physical therapy. Also radiological diagnosis and treatment was not made by single person, hence bias of patient motivation cannot be ruled out.
Only x-rays are used for diagnosis. A huge data suggest intra and inter observer variability in classifications which are based on radiological examination. Hence there is a chance of an overestimate of diagnosis of particular type of fractures.

Conclusions

In this prospective study of 26 patients of stable two part fractures of surgical neck of humerus-
Relevant co-morbid conditions are associated with poor outcome. Four such patients had shoulder stiffness at 6 months with Constant shoulder score <60.
Majority of the patients had angulated two part fractures of surgical neck 50% . Separated group of fracture configuration patients had statistically significant result at 24 weeks.
Constant shoulder score was better at 6 months in two groups and had significant difference at 6 wks and 12 wks.

Referrences

1. Young TB, Wallace WA. Conservative treatment of fractures and fracture dislocations of the upper end of the humerus. J Bone Joint Surg Br. 1985;67:373-7.
2. Clifford PC. Fractures of the neck of the humerus: a review of the late results. Injury. 1980;12:91-5.
3. Kristiansen B, Angermann P, Larsen TK. Functional results following fractures of the proximal humerus. A controlled clinical study comparing two periods of immobilization. Arch Orthop Trauma Surg. 1989;108: 339-41.
4. Hodgson S. Proximal humerus fracture rehabilitation. Clin Orthop Relat Res. 2006;442:131-8.
5. CourtBrown CM, Caesar B. Epidemiology of adult fractures: a review. Injury 2006;37: 691-697.
6. Hodgson SA, Mawson SJ, Stanley D. Rehabilitation after two-part fractures of the neck of the humerus. J Bone Joint Surg Br. 2003;85:419-22.
7. Correard RP, Balatre J, Calcat P. [Results in fractures of the surgical neck of the humerus treated by immediate mobilization. A series of 54 cases in patients over 50]. Ann Chir. 1969;23:1323-6. French.
8. M.M. Lefevre-Colau et al. Immediate Mobilization Compared with Conventional Immobilization for the Impacted Nonoperatively Treated Proximal Humeral Fracture- A Randomized Controlled Trial: J Bone Joint Surg Am. 2007;89:2582-90.

How to Cite this Article: Jangle R D. “Effect of early mobilization in the conservative management of stable two part fracture of surgical neck of humerus”. Journal Medical Thesis 2017 Jan-Apr ; 5(1):18-20.

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A comparative study of management of displaced Gartland Type III supracondylar fracture humerus in children


Vol 5 | Issue 1 | Jan - Apr 2017 | page: 12-17 | Atul A Kharat, Sandeep R Biraris, Nareshkumar S Dhaniwala, Govind J Shinde, Praveenkumar Bhoj


Author: Atul A Kharat [1], Sandeep R Biraris [2], Nareshkumar S Dhaniwala [3], Govind J Shinde [3], Praveenkumar Bhoj [2]

[1] Department of Orthopaedics Pad. Dr. D Y Patil Medical College & Hospital, Nerul, Navi Mumbai, Maharashtra.
[2] Department of Orthopaedics, Mumbai Port Trust Hospital, Wadala (E), Mumbai, Maharashtra.
[3] Department of Orthopaedics S C Medical College, Nanded, Maharashtra.
Institute Where Research Was Conducted: Dr. S. C. Government Medical College, Nanded.
University Affiliation: Maharashtra University of Health Sciences, Nashik.
Year Of Acceptance Of Thesis: 2010.

Address of Correspondence
Dr. Atul A Kharat
Department of Orthopaedics,
Pad. Dr. D Y Patil Medical College & Hospital,
Nerul, Navi Mumbai, Maharashtra.
Email: dratulkharat@gmail.com


Abstract

Background: This study was undertaken to evaluate and compare clinico-radiologically the results of two most common methods of treatment namely, closed reduction and immobilization in plaster slab versus closed reduction and percutaneous K wire fixation and also to study the complications of the management
Materials and Methods: The study “a comparative study of management of displaced Gartland type III supracondylar fracture humerus in children (closed reduction and plaster immobilization versus closed reduction and percutaneous pinning)” was carried out during the period of August 2007 to July 2009 in the department of orthopaedics and attached Civil Hospital. The age of the patient was from 4 to 12 yrs, with follow up ranging from 3 months to 1 year. A total of 98 cases were enrolled for this study. 14 cases were lost in follow up and only 84 patients completing the minimum follow up of 3 months were considered for analysis of observations and results. Out of these 84 patients 54 were treated with closed reduction and K wire fixations while remaining 30 were treated with close reduction only and all the patients were immobilsed with above elbow slab.
Results: Patients were followed up at regular intervals and at each follow-up radiographs were done. Flynn's criterion was used for assessing the functional outcome. As per this criteria 88 % cases of CR+ K wire group had excellent to good results while 67 % cases of CR + slab group had excellent to good results. No case of poor result was noted in k wire group. There were few complications such as elbow stiffness and cubitus varus deformity and tightness of compartment post surgery. These were more common with patients with slab
Conclusion: Our study states that K wire fixation is superior over just plaster immobilization in terms of improved functional outcome and lesser complications.
Keywords: Gartland, supracondylar, humerus fracture, children

Thesis question: Is 'K' wire fixation superior over slab application in supracondylar humeral fractures?
Thesis answer: Our study reveals that 'K' wire fixation with slab is superior over just a slab application in supracondylar humeral fractures.


THESIS SUMMARY

Introduction

Supracondylar fracture of humerus is the most common elbow injury seen in children. Fall on outstretched hand is the most common mode of injury, which occurs in children during playing, running and fall from bicycle. Displaced supracondylar fracture humerus in children may be associated with other fractures of upper extremities depending on the mode of injury. An apparently uncomplicated fracture may lead to complication if not treated properly. This fracture occurs in the area where brachial artery and three nerves of upper extremity are in close vicinity [1]. Hence these structures are liable to get injured as a result of displaced fracture. It may be associated with swelling, limb threatening Volkman ischemia and nerve palsies on presentation and may lead to stiffness of elbow and cubitus varus deformity of elbow [2]. Goals of treatment of supracondylar fracture are to achieve an excellent, functional & cosmetic result without complication [3]. Many methods of treatment have evolved in management of supracondylar fracture humerus in children. These are closed reduction and immobilisation in plaster slab, closed reduction and percutaneous kirschner wire fixation, open reduction and internal fixation, Dunlop traction, olecranon traction etc [2].
This study was undertaken to evaluate and compare clinico-radiologically the results of two most common methods of treatment namely, closed reduction and immobilization in plaster slab versus closed reduction and percutaneous k wire fixation.

Aims and Objective

1. To study clinical profile of dislplaced supracondylar fracture humerus.
2. To compare and evaluate results between closed reduction and immobilzation in plaster slab and closed reduction and percutaneous K wire fixation.
3. To study complication of management by closed reduction and immbilzation in plaster slab and closed reduction and percutaneous K wire fixation.

Material and Methods

Type of study: Prospective
Duration: August 2007 to July 2009

Inclusion Criteria
Traumatic suprocondylar fractures with Type III Gartland, patients willing to participate in the study.
The cases were grouped in two as follows.
Group I: Even no cases treated with closed reduction and K wire fixation.
Group II: Odd no of cases treated with closed reduction and plaster slab
Group I 54 cases (37+8*+9**) and Group II 30 cases constitute the material for this study.
The difference in the number of cases in the two groups is due to
* 8 cases of unstable reduction with plaster slab, K- wire stabilization was done and patient included in Group I fixation.
** 9 cases of suspected of compartment syndrome were treated by CR and K wire fixation.

Exclusion Criteria
Type I and Type II Gartland fractures, Pathological fractures, other fracture involving the same extremity, patients not willing to participate in the study were excluded
Patient followed at 1, 3, 6, 9, and 12 months and each follow-up results were assessed using Flynn's criteria [4].

Results

In the present study 98 cases of Gartland type III supracondylar fracture humerus were enrolled. Only 84 cases were taken for final results analysis as they had completed the minimum follow up of 3 months. Out of 14 cases lost in follow up, 11 were in CR+ slab group and 3 were from CR + K wire group. Out of 84 cases considered for results 54 belonged to CR + K wire group and 30 cases were of CR+ slab group. The following observations of the study are based on the follow up of 84 cases only. Around 72% patients were between 5 to 10 years of age and 82% were boys. Left side injury was 3 times commoner than right. Fall on outstretched hand was common mechanism of injury (60%). Posteromedial displacement was 5 times commoner than posterolateral. 5 patients had associated distal radius fracture while vascular compromise was seen in 7 patients and one patient had nerve injury. 81% patients were treated with lateral 'K' wire fixation. 76.19% patients were having metaphyseo-diaphyeal angle between 85-100 postoperatively, indicating good reduction of fracture. However, values less than 85 and above 100 in a total of 20 cases were accepted. It was found that incidence of stiff elbow and cubitus varus was more common in patients treated with slab group (6.66%). Around 87.03 % cases treated with 'K' wire had less than 10-degree loss of range of motion. While this percentage was 56.66 % in cases treated with plaster slab alone. 6.66% (2) cases managed with slab had cubitus varus while none case of K wire group had cubitus varus. Thus showing overall results are better in 'K' wire group. As per Flynn's criteria 88 % cases of CR+ K wire group had excellent to good results while 67 % cases of CR + slab group had excellent to good results. No case of poor result was noted in k wire group while 2 patients in slab group had poor outcome.

Discussion

The average age incidence in the present study is consistent with other studies and shows that supracondylar fracture of humerus of immature skeleton is common during the end of first decade. This was consistent with other studies. Many studies have shown incidence of fracture higher in boys than girls same observations were made in our study. Involved site in literature was non-dominant side it was same in our study as well. Fall over outstretched hand was more common mechanism of injury. Palmer et. al (1978) in series of 78 patients of supracondylar fractures of the humerus showed 5.2% (4 cases) were having ipsilateral fracture of the lower end of the radius [5]. In 1988, Pirone and Graham in their series found 9% of cases of other ipsilateral fractures[6]. In the present study of 84 cases, 5.95% (5 cases) patients were associated with ipsilateral fracture of distal end radius. Pirone AM et al (1988) observed posteromedial displacement in 68.61% cases, posterolateral in 16.05% cases and directly posterior displacement in 15.32% cases [6].In the present study of 84 cases, 80.9% (68 cases) had posteromedial displacement, 16.6 %( 14 cases) had postero lateral displaced and 2.38 % (2 cases) anterior displacement. Posteromedial : posterolateral = 5:1
Conclusion
This study shows supracondylar fracture humerus to be more common in boys between 5-10 years' age, more on nondominant side and has posteromedial displacement.
The study found that better maintaince of reduction by K wire fixation with better follow up and avoidance of complications of compartment syndrome, stiffness and cubitus varus in comparison to plaster slab.

Clinical Message

This study tells that supracondylar fracture humerus is seen most commonly in non-dominant hand, most common age group is 5-10 years. K wire fixation with immobilization has better functional outcome than only immobilsation.

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How to Cite this Article: Kharat A A, Biraris S R, Dhaniwala N S, Shinde G J, Bhoj P. A comparative study of management of displaced Gartland Type III supracondylar fracture humerus in children. Journal Medical Thesis 2017 Jan-Apr ; 5(1):12-17.

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“Gas Chromatography Mass Spectrometry Determination of Alcohol in Herbal Medicines Available to the Herbalists”


Vol 5 | Issue 1 | Jan - Apr 2017 | page: 6-11 | Rumi Dasgupta


Author: Rumi Dasgupta [1]

[1] University of East London, Stratford, London.
Institute Where Research Was Conducted: School of Health, Sports and Biosciences, University of East London, Stratford, London.
University Affiliation: University of East London.
Year Of Acceptance Of Thesis: 2009.

Address of Correspondence
Dr. Rumi Dasgupta
Research Trainee, MS (Toxicology), University of East London,
Stratford, London.
Email: rumidasgupta@gmail.com


Abstract

Background: Herbal medicine is the most widely accepted system of medicine used for curing and prevention of various diseases. People all over the globe use herbal medicines. Due to its acceptability by people, as these medicines are made from herbs, the safety, efficacy and clinical trials of herbal medicines are generally not carried out. The herbal tinctures which are prescribed by the qualified herbalists specify the amount of alcohol present in the tincture. Alcohol is used as a solvent for making tinctures. This study aims to analyse the percentage of alcohol present in the herbal tinctures as specified in the labels.
Method: To carry out the analysis gas chromatography with flame ionisation detector has been used with a ZB – Wax Plus wax column. The method is simple and sensitive.
Results: The results suggest that herbal tinctures of Melissa officinalis which claims to have an alcoholic content of 25%, 30%, 40%, 45%, 66% and 70% contains higher amount of ethanol in it than it is prescribed in the labels. It suggests Melissa officinalis containing 66% of alcohol contains 88% of ethanol and 0.09 ppm of methanol in it. The ethanol content is lower than it is prescribed in the labels of the herbal tinctures of Chamomile. A very high amount of methanol, i.e. 1.9 ppm of methanol is present in herbal tincture of Chamomile containing 45% of alcohol.
Conclusion: Due to the presence of different active components in the herbal tinctures, it may be possible that the peaks obtained can be of some of those active components present in the herbal tincture. It is also possible that these active components are overlapping with the peaks of methanol and ethanol. It is difficult to segregate the peaks of active components which are overlapping with that of methanol and ethanol peaks. To confirm it, further analysis is required, which can be carried out by using Gas Chromatography Mass Spectrometer Method or HPLC technique.
Keywords: Herbal medicines, Alcohol, Gas Chromatography, Flame Ionisation Detector.
Thesis Question: Is the ethanol content of the Melissa officinalis and Matricaria reticutita medicines prescribed by the herbalists like the content prescribed in the label?
Thesis Answer: The amount of ethanol varies with tincture preparation and active compounds present. The peaks also suggest the presence of methanol content in the tinctures. The amount higher than 1 ppm suggests that there is a significant amount of methanol present..


THESIS SUMMARY

Introduction

Herbal medicine is the most common and widely accepted system of medicine. It is due to its natural nature. Herbal practioners prescribe these medicines to their patients. Because of its nature, herbal medicines are sold in the market without conducting tests for safety, efficacy and clinical trials, unlike the pharmaceutical drugs[1]. Thus, many plants which are poisonous in nature affect the patient adversely by triggering adverse reactions in the body. Alcohol is used as a solvent for the preparation of herbal medicines. It has been observed that the content and amount of the content is generally not mentioned in the labels of the herbal medicines. The aim of this project is to test the amount of alcohol present in the herbal medicines which is prescribed by the herbalists, mentioned in the labels and to identify the contaminants present in the herbal medicines, if any, by using gas chromatography flame ionisation detector technique. Herbal medicines which will be used for the analysis are generally prescribed by the herbal practioners and taken by the people. Gas chromatography with flame ionization detector is a definitive technique, hence it will be used to separate present in the herbal medicines along with the percentage of alcohol present in the herbal medicines.
There are several systems of herbal medicine which governs the practice of herbal medicine. They are: Chinese Herbal Medicine or Chinese Herbology, Siddha and Ayurvedic systems from South Asian Countries, Herbal medicine system based on Roman and Greek sources, Unani – Tibb medicine and Shamanic herbalism. There are diverse types of herbal medicines which are used across the globe for the treatment of various diseases. The common types of medicinal uses are herbal teas, herbal tinctures, fluid & solid extracts, poultices and essential oils[2].
Traditionally, herbal medicines are prepared in standardized ways, which varies as per the plants utilized and the condition they are being treated. The standardized methods include infusions, decoctions, tinctures and macerations. These tinctures are prescribed by the herbal practioners to their patients.
Herbal alcoholic tinctures are usually used herbal medicines. Generally, tinctures are easily taken up by the body than water – soluble teas. Due to the presence of alcohol, tinctures can be preserved as compared to herbal teas or water soluble herbal medicines. In general, alcohol is a better solvent than water as the plant constituents dissolve in alcohol more readily than water.
Tinctures are the most commonly used methods for treatment. It is a fluid extract prepared from the herbs[1]. A tincture is a water and alcohol extract, which is used when plants have active chemicals which are not very water soluble. The alcoholic percentage helps to determine the shelf – life of a tincture. Alcoholic percentage or content is directly proportional to the shelf – life of tincture. Alcoholic percentage differs from plant to plant as some active compounds have higher affinity for alcohol while some have higher affinity for water. Mostly vodka, rum or 90 – 180 proof grain alcohols is used for the preparation of tinctures[3].
A standard 4:1 tincture indicates 4 parts of liquid and 1 part of plant part used. One year shelf – life tincture can be prepared by using 40% alcohol, i.e. 80 proof vodka or rum without water. Approximately a cup of tincture can be prepared by mixing two ounces of herbs, four ounces of distilled water and four ounces of 180 proof alcohols into a container. Once the mixing is done, the container is sealed and stored at room temperature for about four weeks. After two weeks, the mixture is filtered to remove the plant parts and then pouring the tincture in a cleaned container and sealing it. This technique uses a higher plant to liquid ratio; the tinctures are usually lower than infusions and decoctions.
Herbs depend on its bioavailability factor as they are introduced in the body through topical or oral routes, unlike the pharmaceutical drugs which can be directly introduced in the blood stream[3][4]. Bioavailability plays an important role in delivering the dose of active compounds as it defines the amount of active substances absorbed into the bloodstream after oral doses.

Aims & Objectives

The aim of my project, as a toxicologist, is to analyse the percentage of alcohol present in the herbal medicines available to the herbalists as prescribed in the labels. These herbal medicines are generally prescribed by the qualified herbal practioners to their patients. The analysis will be done by using gas chromatography with flame ionization detector instrument. This technique has been chosen because it is a definitive technique and can easily identify the components present in the herbal medicines[4].
This experiment helped in the analysis of the amount of percentage which is present in the herbal tincture as claimed by the manufacturers and to check if there are any impurities like methanol, iso – butanol, propan – 1 – ol, etc. present. Since tests for safety, efficacy and clinical trials are not carried out for herbal medicines, this experiment is helping us to assess the authenticity of the content which is claimed by the manufacturers.
This was carried out looking for a suitable solvent for the analysis and an internal standard which has a different retention time than methanol and ethanol. An internal standard is required for the comparison of the data and to validate the results. The calibration of methanol and ethanol are then carried out. Methanol calibration was done because methanol is the commonly present impurity and ethanol calibration is done because ethanol is used as a solvent for the herbal extracts or medicines.
Once the calibration was obtained different samples were taken and diluted as per the requirement and were run on GC. The data obtained gave us the peak area and the retention time helped to sort out the methanol, ethanol and the acetonitrile (the internal standard) from the chromatogram obtained after the analysis. Further calculation of the percentage of alcohol present was carried out by comparing the peak area and the calibration curve of methanol and ethanol.

Materials and Methods

Materials which will be used for analysis are the herbal medicines which are generally prescribed by the herbalists. The herbal medicines which are going to be used for this project are Lemon Balm (Melissa officinalis) and Chamomile (Matricaria reticutita). For the analysis, herbal medicines which are generally prescribed by the qualified herbalists have been used[5][6].
Gas chromatography flame ionisation detector is a technique which is used to separate the compounds present in the herbal medicine and identifies the compounds which has been separated at a molecular level[6]. Gas chromatography works on the separation principle where components present in the herbal medicine will get separated into individual substances when excited. The excited gases are carried through a column with an inert gas. As the separated substances emerge from the column opening, they flow into mass spectrometer. Gas chromatography has many advantages like it can detect volatile compounds that are present in the sample. It requires less time to carry out the analysis and the results are obtained within 1 – 100 minutes. This technique is good for the separation of different components present in the sample. The precision of gas chromatography is very high. The equipment is simple to handle[6][7][8].
This method will be used to determine the alcoholic content of the herbal medicines as it gives accurate results. It is a definitive tool; hence this method is chosen to carry out this experiment. These experiments will help to analyse how much percentage of alcohol the herbal medicine contains and if there is any impurity present. Since tests for safety, efficacy and clinical trials are not conducted for herbal medicines, this experiment will help to assess the authenticity of the information provided in the labels of the herbal medicines and safety of the herbal medicines[9][10][11].
Gas Chromatography Flame Ionisation Detector which has been used for the analysis is from the Agilent Technologies 7890A GC System with a ZB – Wax Plus wax column. The length of the column which has been used was 30 m, the internal diameter was 0.25 mm and the thickness of the film was 0.25 µm. The temperature range was 20ᵒC (minimum) to 250ᵒC (maximum).
To choose a solvent for the dilutions, absolute methanol and ethanol was run by diluting the chemicals in water. Before running the samples, water was run as a blank to eliminate the presence of unwanted or leftover organic compounds. The standards for methanol and ethanol was made by using absolute methanol and ethanol and diluting it to 1:10 ratio for methanol and 1:1000 ratio for ethanol by using distilled water. To make 1:10 dilution for methanol and 1:1000 dilutions for ethanol, following calculations were used:
C1V1 = C2V2
Where,
C1= Concentration of the stock
V1= Volume of the stock
C2= Concentration of the solution
V2= Volume of the solution
Different concentrations were made by using the above diluted solutions. To it the 1:10 diluted acetonitrile was added as acetonitrile was used as an internal standard. The standards were then run and readings were recorded.
The samples of medicines with different alcoholic content were then taken and a dilution of 1:10 ratio and 1:1000 ratio dilutions were made for methanol and ethanol analysis respectively. Then the samples were centrifuged to get a clear solution as particulate matters were present. After centrifuging the internal standard, i.e. acetonitrile was added and then the samples were run. The observations were then recorded.
A range of concentration ranging from 0 – 5 ppm were made for 1:10 dilution of methanol calibration curve and 0 – 100% were made for 1:1000 dilution of ethanol calibration curve. The analysis will be carried out by comparing the concentrations in the calibration curve to determine the alcoholic content of the herbal medicines[10][11].
Acetonitrile was used as an internal standard as the retention time of acetonitrile is different from that of methanol & ethanol and it does not overlap with the retention time of methanol and ethanol[4][12][13][14]. A 1:10 dilution of acetonitrile was used for the analysis of the samples. 500 µl of acetonitrile was taken in a 5-ml volumetric flask and then distilled water was added till the mark to make 5 ml of 1:10 dilution of acetonitrile solution[4][5][15][16].
The results in Table 1 give us the calibration for methanol and results in Table 2 is for the calibration of ethanol. The calibration was carried out by making a dilution of 1:10 for methanol and 1:1000 for ethanol. A dilution of 1:10 was carried out for methanol because the concentration of methanol in the solution is present in trace amounts. To detect those trace amounts of methanol a 1:10 dilution was carried out for methanol. Ethanol, on the other hand, gives a very sharp peak and hence a dilution of 1:1000 was carried out for ethanol.

Results

The retention time of acetonitrile was 7.16 mins, ethanol was 5.82
mins and methanol was 4.89 mins. Once the chromatograms were obtained integration method was used for the quantitation or the values of the peaks. The parameters were then set.
Once the parameters were set the APPLY option was clicked and then OK was clicked. This gave the retention time of the peaks and the peak areas which helped in the determination of the content of the alcohol present in the herbal tincture samples.
The results were obtained from the calibration curve. The results of the analysis are as follows:
1. Ethanol Content of Melissa officinalis: (Table 1)
CONCENTRATION AS PRESCRIBED ETHANOL CONTENT (%)
25% Melissa 52
30% Melissa 48
40% Melissa 72
45% Melissa 82
66% Melissa 88
70% Melissa 79

2. Methanol Content of Melissa officinalis: (Table 2)
CONCENTRATION AS PRESCRIBED METHANOL CONTENT (ppm)
25% Melissa 0.1
30% Melissa 0.3
40% Melissa 0.1
45% Melissa 1.7
66% Melissa 0.09
70% Melissa 0.2

3. Ethanol Content of Chamomile: (Table 3)
CONCENTRATION AS PRESCRIBED ETHANOL CONTENT (%)
45% Chamomile 34
60% Chamomile 2
66% Chamomile 18

4. Methanol Content of Chamomile: (Table 4)
CONCENTRATION AS PRESCRIBED METHANOL CONTENT (ppm)
45% Chamomile 1.9
60% Chamomile 0.6
66% Chamomile 0.1.

Observations

The standard calibration curve for ethanol suggests that there may
be some error while preparing the concentrations. The calibration curve of methanol shows a straight line with R2 = 0.990. It suggests that the preparation of concentrations of methanol calibration curve was fine with not much error. The standard calibration curve is made by diluting the sample with water.
The results in Table 1 suggest that herbal tinctures of Melissa officinalis which claims to have an alcoholic content of 25%, 30%, 40%, 45%, 66% and 70% contains higher amount of ethanol in it than it is prescribed in the labels. This suggests either there is a more number of active components of plants are present for which more ethanol has been used to make it soluble or maybe there was some fault in the Gas Chromatography instrumentation. The above table suggests Melissa officinalis containing 66% of alcohol contains 88% of ethanol and 0.09 ppm of methanol in it.
The percentage of alcohol present in the sample of Melissa officinalis against response or ethanol peak area with ethanol content is 48% - 88% suggests that ethanol content is more in the herbal tincture of Melissa officinalis with 66% of alcohol (88%). The herbal tincture with 30% of alcohol has lesser ethanol content (48%) as compared to the others, though the overall results show increased ethanol content in the tinctures with different alcoholic content. Figure 4 shows the chromatogram of ethanol content in the sample of Melissa officinalis with 66% of alcohol content as prescribed in its label.
The comparison of the methanol content in different samples of Melissa officinalis with methanol content range lies between 0.07 – 0.3 ppm suggests that the tincture with 70% alcohol has more amount of methanol (0.3 ppm) in it and the tincture with 25% of alcohol has the lowest methanol content (0.07 ppm).
The percentage of alcohol present in the sample of Chamomile in a range of ethanol content in Chamomile lies between 18 – 34%. It suggests that the tincture with 45% of alcohol has more ethanol content (34%) and the tincture with 66% of alcohol has fewer amounts (i.e. 18%) of ethanol in it.
The percentage of alcohol present in the sample of Chamomile against the methanol peak area with methanol content ranges between 0.1 – 1.9 ppm suggests that the tincture with 45% of alcohol has 1.9 ppm of methanol and the tincture of Chamomile with 66% of alcohol contains 0.1 ppm of methanol.
It also suggest that the ethanol content is lower than it is prescribed in the labels of the herbal tinctures of Chamomile. This suggests that maybe the medicine has less active compounds present in it and has a placebo effect on the patients or due to the breakdown of Gas Chromatography Instrumentation the results obtained were not accurate. A very high amount of methanol, i.e. 1.9 ppm of methanol is present in herbal tincture of Chamomile containing 45% of alcohol. Generally, 1 ppm is the maximum level till which methanol can be present. The amount higher than 1 ppm suggests that there is a significant amount of methanol is present. Methanol is a common adulterant present in the herbal tinctures, as tinctures contain alcohol as a solvent.
Conclusion
The accuracy of the results is not very high. It is due to the breakdown of the instrument and maybe error was there while making the dilutions. The calibration curve was carried out without the addition of the internal standard. The second sets of reading could not be taken due to the breakdown of the instrumentation. A further analysis is required to confirm the results and identify the different peaks that were found during the analysis as Gas Chromatography Flame Ionisation Technique detects any organic components present in the sample but a Mass Spectrometer or HPLC system is required for the identification of all the organic compounds detected by the Gas Chromatography.
Further work needs to be done for the confirmation of the results. HPLC and/or a Mass Spectrometer can be used to identify the different components of the herbal tinctures. Since the Gas Chromatography Flame Ionisation Detector cannot reveal the molecular formula of the components, hence one can be never be sure if the peak is for methanol and ethanol exclusively. Due to the presence of different active components in the herbal tinctures, it may be possible that the peaks obtained can be of some of those active components present in the herbal tincture. It is also possible that these active components are overlapping with the peaks of methanol and ethanol. It is difficult to segregate the peaks of active components which are overlapping with that of methanol and ethanol peaks. To confirm it, further analysis is required, which can be carried out by using Gas Chromatography Mass Spectrometer Method or HPLC technique.

References

1. Mills, S. & Bone, K. (2001) Principles and Practice of Phytotherapy: Modern Herbal Medicine, Churchill Livingstone
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How to Cite this Article: Dasgupta R. “Gas Chromatography Mass Spectrometry Determination of Alcohol in Herbal Medicines Available to the Herbalists”. Journal Medical Thesis 2017 Jan-Apr ; 5(1):6-11.

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Early Intervention with Muscle Energy Technique has better improvement in pain, range of motion and function in post surgical elbow stiffness: A hypothesis


Vol 5 | Issue 1 | Jan - Apr 2017 | page: 3-5 | Anood I Faqih, Nilima S Bedekar


Author: Anood I Faqih [1], Nilima S Bedekar [1]

[1] Department of Musculoskeletal Physiotherapy, Sancheti Healthcare Academy, Sancheti Institute College of Physiotherapy, Thube Park, Shivaji Nagar, Pune, Maharashtra, India.
Institute Where Research Was Conducted: Sancheti Institute of Orthopaedics and Rehabilitation.
University Affiliation: Research Hypothesis (Synopsis) submitted for MPTh Registration to Maharashtra University of Health Sciences (MUHS), Nashik.
Year Of Acceptance Of Thesis: 2016

Address of Correspondence
Dr. Nilima S Bedekar
Sancheti Healthcare Academy, Sancheti Institute College of Physiotherapy, 12, Thube Park, Shivaji Nagar, Pune - 411005, Maharashtra.
Email: nilimabedekar@yahoo.com


Abstract

Background: Stiffness is common post trauma and post surgeries around the elbow. This could result in functional disabilities in the upperlimb and may interfere with activities of daily living in the individual. Also, post operative pain is a contributing factor to these disabilities. Optimal joint mobility is essential to overcome stiffness and hence improve the function. Muscle energy techniques (MET) have been used in the treatment of restricted joint mobility to allow relatively pain free range of movement. Hence, the purpose of this study is to evaluate the effects of MET on pain, ROM and function post elbow surgeries in population aged between 18-50 years by an experimental study on 30 participants divided in 2 groups (15 each) in which MET will be given to group A immediately as per the current literature and to group B after 1 week as per the protocol of the institution .The effect of MET given per week will be assessed in a period of 3 weeks.
Hypothesis: The group in which MET will be started immediately post immobilisation will show better improvement in pain, ROM and function in patients with post surgical elbow stiffness.
Clinical Importance: Adding muscle energy technique to the elbow programme early would show better improvement in pain and elbow ROM and in turn improve the function of the upper extremity.
Future direction: To study the long term effects of Muscle energy technique in post surgical elbow stiffness.
Keywords: Muscle Energy Technique, Elbow Stiffness.


THESIS SUMMARY

Introduction

Restriction of joint mobility is a common complication that is seen post elbow surgery [1]. This could be due to immobilization, pain, muscle guarding etc. All these may lead to reduced joint function and may hamper the patient’s ability to perform functional tasks, thereby affecting his activities of daily living [2][3].
Muscle energy techniques (MET) are soft tissue or joint techniques which are employed in the treatment of musculoskeletal dysfunctions. Post operative pain is one of the factors that reduce the patient’s compliance and does not allow optimal joint and muscle mobilization. Also, passive rehabilitation techniques may cause adverse effects to the fragile tissues in the post operative period in elbow joint. METs are a group of relatively pain free mobilization techniques which are used to regain mobility, reduce tissue edema, reduce muscle spasm, stretch fibrous tissue and retrain stabilizing function of the intersegmentally connected muscles [4].
The elbow being a highly constrained synovial hinge joint has a high propensity for degeneration and stiffness. Elbow motion is required to position the hand in space. There could be functional losses seen with even less severe loss of range of motion at the elbow. A stiff elbow has been defined as the one with loss of extension of greater than 30 degrees and flexion of less than 120 degrees [5].
Elbow stiffness could arise due to various reasons, trauma being the most common cause. There can be voluntary or involuntary muscle guarding of the elbow during motion due to prolonged pain. This could lead to contractures in the elbow joint capsule and also to the muscles around it [5]. Contractures which may develop post trauma can impair activities of daily living and may also cause functional limitations in children and adults [6]. There is poor literature on the use of Muscle energy techniques in rehabilitation post elbow surgeries. Elbow is a very functional joint in the upper extremity. An arc of motion of 30-130 degrees is required to carry out activities of daily living. Elbow stiffness is a significant cause of disability which may hamper the function of the upper extremity as a whole. METs are relatively painfree techniques which have been used in clinical practice for restricted ROM. Hence, the purpose of this study is to know the effect of MET on pain, ROM and function in patients with post surgical elbow stiffness.

Hypothesis

The current study aims at studying the effects of MET on pain, elbow ROM and function in patients with post surgical elbow stiffness in participants aged 18 to 50 years. This is an experimental study which will be performed after ethical clearance from the institute’s ethical committee. The sampling method will be a stratified random using the chit method after obtaining an informed consent from the participants. Patients with post operative distal end extra-articular or intra-articular humerus fractures and/or proximal radius ulna fractures without any ligament injury with a minimum immobilization period of 3 weeks will be included in the study. Those having pathological fractures, revision surgeries, associated ipsilateral injuries and neuro-vascular disorders will not be included in the study.
For pain evaluation, Visual Analog Scale will be used. For Range of Motion, a goniometer will be used and to assess the function the Disability of arm, shoulder and hand (DASH) questionnaire will be used. Pain, ROM and function will be assessed before starting the treatment and at the end of every week for 3 weeks.
The participants will be assigned to 2 groups: Group A will receive active and active-assisted ROM exercises consisting of elbow flexion-extension, pronation-supination, active exercises for the wrist and shoulder and gentle passive stretching and MET in the form of post isometric relaxation and/or reciprocal inhibition immediately. Group B will receive the same treatment but MET will be started after 1 week.
The subjects will be assessed separately for intra-articular, extra-articular, simple and compound type of fractures. For ROM within group and between group analysis, one way ANOVA will be used. For pain and function within group Friedman test will be used and between both the groups Kruskalwallis test will be used.

Discussion

The present study will be undertaken to study the effects of Muscle energy technique when applied immediately and after 1 week of elbow rehabilitation on pain, elbow ROM and function in patients with post surgical elbow stiffness.
The group in which MET will be started immediately will show better improvement in the parameters mentioned above. The reduction in pain intensity in the groups would be attributed to the hypoalgesic effects of MET which is explained by the inhibitory Golgi tendon reflex, activated during the isometric contraction that in turn leads to the reflex relaxation of the muscles. Also, the muscle and joint mechanoreceptors would be activated leading to leads to sympatho-excitation evoked by somatic afferents and localized activation of the periaqueductal gray matter. This plays a role in the descending modulation of pain.
Also, the hypomobility associated with reflex muscle guarding due to pain would reduce as pain reduces.
MET can be used to improve joint ROM and has an advantage over standard stretching techniques to gain early ROM in post surgically treated fracture cases.[4]
A study done by Gupta.S(2008) on effects of post isometric relaxation versus isometric exercises in non specific neck pain also concluded that MET showed significant improvement in pain and functional status. Convenient sample of 37 non specific neck pain patients between the age group 18-45 years were allocated randomly to each group. Both the groups received the selective treatment for 3 weeks. Pain (VAS), ROM and function was measured with the Neck Disability Index on the 1st, 8th, 15th and 22nd day.[8]
A.Shyam, S.Parmar(2011) did a case study on a 50 year old lady with left supracondylar fracture humerus managed surgically with 2 screws and a plate 7 weeks post operatively. MET was given for 8-10 repititions once a day for 6 days a week for 40 sessions after which the elbow ROM improved significantly from 30-70 degrees of flexion to 0-135 degrees of flexion and from 10 degrees of pronation to 90 degrees and 50 degrees of supination to 90 degrees. Also, a similar case study was done by them on a 48 year old male diagnosed with closed fracture right humerus with elbow link protocol 3 weeks post operatively. MET was given for 8-10 repititions once a day for 6 days a week for 30 sessions after which the elbow ROM improved significantly from 40-90 degrees of flexion to 0-140 degrees of flexion and from 30 degrees of pronation to 90 degrees and 25 degrees of supination to 90 degrees.[4]
Our results of ROM improvement could be supported with a study done by Stephanie M in 2011 titled the immediate effects of muscle energy technique on post shoulder tightness in which they concluded that a single application of MET provides significant improvement in shoulder adduction and internal rotation ROM.[9]
Davila SA, Johnston-Jones K(2006) in their study, “Managing the stiff elbow: operative, nonoperative and post-operative techniques” stated muscle energy technique, active range of motion exercises as one of the many treatment techniques for managing the stiff elbow.[10]
Apoorva Phadke, Nilima Bedekar(2016) in their study,”Effect of muscle energy technique and static stretching on pain and functional disability in patients with mechanical neck pain: A randomized control trial” concluded that muscle energy technique was better than stretching technique in improving pain and functional disability in people with mechanical neck pain.[11]
Active ROM, Active- Assisted ROM, passive ROM and stretching would help in improving the range of motion. This is supported by Joy C MacDermid et al(2015) in their study “A Survey of Practice Patterns for Rehabilitation Post Elbow Fracture” which concluded that active ROM exercises, active-assisted ROM exercises, passive ROM exercises and stretching have high consensus as components in the rehabilitation post elbow fractures.[12]
MET will show better improvement in elbow range of motion. This could be explained by the hypothesis suggested by by Taylor et al in their study done in 1997, suggested that a combination of contractions and stretches (as used in MET) might be more effective in producing viscoelastic changes than passive stretching alone, because the greater forces produce increased viscoelastic change and passive extensibility.[11][13]
Lendermanin 1997 proposed that passive stretching would elongate the parallel fibers but have little effect on the ‘in series’ fibers; however, the addition of an isometric contraction would place loading on these fibers to produce viscoelastic or plastic changes above and beyond that achieved by passive stretching alone.[14]Active muscle contraction has been shown to have neurophysiological effects, including pain inhibition, thus allowing the muscles to be stretched further .[11][15]
Hence, early intervention with muscle energy technique will show better improvement in pain, range of motion and function in patients with elbow stiffness post surgery.

References

1. Muller AM, Sadoghi P, Lucas R, Audige L, Delaney R, Klein M, Valderrabano V, Vavken P. Effectiveness of bracing in the treatment of nonosseous restriction of elbow mobility: a systematic review and meta-analysis of 13 studies . J Shoulder Elbow Surg. 2013 Aug;22(8):1146-52.
2. Parmar S, Shyam A. The effect of isolytic contraction and passive manual stretching on pain and knee range of motion after hip surgery: A prospective, double-blinded, randomized study. Hong Kong Physiotherapy Journalc2011; 29:25-30.
3. Kisner C, Colby L. Therapeutic exercises foundation and techniques. 4th ed. Bangalore, India: Jaypee Brothers; 2001.p.171e80.
4. Chaitow L. Muscle energy techniques. 4th ed. Edinburgh: Churchill Livingstone 2013:94,243,245,249,250.
5. Nandi S, Maschke S, Evans PJ, Lawton JN. The stiff elbow. Hand (N Y). 2009Dec;4(4):368-79. doi: 10.1007/s11552-009-9181-z. Epub 2009 Apr 7. PubMed PMID:19350328; PubMed Central PMCID: PMC2787214.
6. Bruno RJ, Lee ML, Strauch RJ, Rosenwasser MP. Posttraumatic elbow stiffness:evaluation and management. J Am Acad Orthop Surg. 2002 Mar-Apr;10(2):106-16.PubMed PMID: 11929205.
7. Jandric S, I Sar. Functional outcome of posttraumatic elbow contracture. Med Pregl.2010 Jul-Aug;63(7-8):546-9.
8. Gupta S, Jaiswal P. A comparative study between post isometric relaxation and isometric exercises in non-specific neck pain. Journal of exercise science and physiotherapy 2008; 4:88-94.
9. Moore SD, Laudner KG, McLoda TA, Shaffer MA. The immediate effects of muscle energy technique on posterior shoulder tightness: a randomized controlled trial. J Orthop Sports Phys Ther. 2011 Jun;41(6):400-7. doi: 10.2519/jospt.2011.3292.Epub 2011 Apr 6. PubMed PMID: 21471651.
10. Davila SA, Johnston-Jones K. Managing the stiff elbow: operative, nonoperative and postoperative techniques. J Hand Ther. 2006 Apr-Jun;19(2):268-81.
11. Phadke A, Bedekar N, Shyam A, Sancheti P. Effect of muscle energy technique and static stretching on pain and functional disability in patients with mechanical neck pain: A randomized controlled trial. Hong Kong Physiotherapy Journal 2016; 35:5-11.
12. Macdermid JC, Vincent JI, Kieffer L, Kieffer A, Demaiter J, Macintosh S. A survey of practice patterns for rehabilitation post elbow
fracture. Open Orthop J. 2012;6:429-39.
13. Taylor DC, Brooks DE, Ryan JB. Viscoelastic characteristics of muscle: passive stretching versus muscular contractions. Med Sci Sports Exerc. 1997Dec;29(12):1619-24. PubMed PMID: 9432095.
14. Lenderman E (1997).Fundamentals of manual therapy.Churchill Livingstone. London; cited by Chaitow L. Muscle energy techniques.3rd ed. Edinburgh: Churchill Livingstone 2006:122.
15. Ylinen J. Stretching therapy for sports and manual therapies. Section 1d stretching theory. 1st ed. Oxford, United Kingdom: Churchill Livingstone; 2008. p. 22-102.

How to Cite this Article: Faqih A I, Bedekar N. Early Intervention with Muscle Energy Technique has better improvement in pain, range of motion and function in post surgical elbow stiffness: A hypothesis. Journal Medical Thesis 2017 Jan-Apr ; 5(1):3-5.

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“A Clinical Study of Functional Outcome After Management of Supracondylar Fracture Humerus in A Child”


Vol 5 | Issue 1 | Jan - Apr 2017 | page: 27-31 | Vibhute Nilesh Deepakrao, Roney Thomas, Tony Kavalakkatt


Author: Vibhute Nilesh Deepakrao [1], Roney Thomas [1], Tony Kavalakkatt [1]

[1] Baby Memorial Hospital, Calicut, Kerala.
Institute Where Research Was Conducted: Baby Memorial Hospital, Calicut, Kerala.
University Affiliation: National Board of Examinations, New Delhi.
Year Of Acceptance Of Thesis: 2013.

Address of Correspondence
Dr. Vibhute Nilesh Deepakrao
Baby Memorial Hospital, Calicut. Kerala.
Email: dr.nileshvibhute03@gmail.com


Abstract

Objectives: Functional outcome following management of supracondylar Humerus fracture in a child was evaluated with different methods of management.
Method: 37 cases of supracondylar fractures treated by various methods [including conservative & operative methods] were studied between April 2011 to May 2013 at our institution and followed for an average of 6 months. Patients were treated with Closed reduction K wire fixation (open reduction if required) or closed reduction and strapping, or conservatively.
Results: Total 37 patients were evaluated. Out of 20 patients with TYPE III fracture treated by K wire fixation 13 (65%) patients had excellent results, 6 (30%) patients had good results & 1(5%) patient had fair result. The remaining 10 (33.33%) patients with type III fracture was treated with closed manipulation & reduction & above elbow pop slab and strapping of elbow around chest. , out of which 6 (60%) patient had excellent results, 2 (20%) patients had good results, 1 (10%) patient had fair result & 1(10%) patient had poor results. Out of 5 patients with type II fracture, 4 (80%) patients were treated conservatively with closed reduction and above elbow pop slab & all had excellent results, 1(20%) patient was treated with open reduction & internal fixation with k wires had good result. Out of 2 patients with TYPE I fracture all were treated conservatively in an above elbow pop slab & all had excellent results according to Flynn's criteria.
Conclusion: The results of Conservative treatment with closed manipulation & reduction with above elbow pop slab (elbow in 1100-1200 flexion) application and strapping of elbow around chest in type III fracture supracondylar fractures of humerus in children are almost similar to the operative group but there is high chance of increased rate of complications like loss of reduction, malunion & restriction of movement especially if it's done in older age group children.
Keywords: Supracondylar fracture Humerus, Distal humeral fractures.


THESIS SUMMARY

Introduction

Supracondylar fracture of humerus is the commonest injury around elbow in children. It constitutes about 65.4% of all the fractures about the elbow in children. Although the bony architecture of the distal humerus is responsible for the frequency of supracondylar humeral fractures, it is the soft tissue anatomy that has the potential to produce devastating long-term complications.
The management of displaced Supracondylar fracture of the humerus is one of the most challenging one to prevent complications. No single method of management is suitable for all Supracondylar fractures in children.
There is no controversy regarding treatment of undisplaced supracondylar fractures. But various modalities of treatment have been proposed for the treatment of displaced supracondylar fractures of the humerus in children.

Aim of Study

To study the Age, Sex and Side incidence of supracondylar fractures of humerus in children below 14ys of age.
To know the most common mechanism of injury.
To study complications associated with it.
To study outcome of conservative management with POP application & elbow strapping.
To study outcome of surgical management.
Percutaneous K wire fixation (Closed method)
Open reduction and k wire fixation

Methodology

37 cases of supracondylar fractures treated by various methods [including conservative & operative methods] were studied between April 2011 to May 2013 at our institution and followed for an average of 6 months.
All children upto 14 years of age with supracondylar fractures of humerus Closed fractures were included. Children more than 14yrs of age were excluded. The patients with open fractures and other fractures of the same extremity or polytrauma were also excluded from study.
The ethical clearance for this study has been taken from our institution. All patients selected for this study were admitted in Baby Memorial Hospital, Calicut Kerala and were examined according to protocol.
Preoperative informed consent was taken from the parents of the patient for anaesthesia and procedure.
We studied total of 37 patients out of which 30 were type III fractures, 5 type II fractures and 2 type I fractures.
Out of 30 patients with type III fracture, 20 patients were treated with surgical fixation which included 16 percutaneous pinning and 4 open reduction and k wire fixation.
Closed reduction was done under general anesthesia with use of image intensifier. Firstly, traction is applied with the elbow in 45o flexion and forearm in supination. While the traction is maintained the medial or lateral displacement is corrected by applying a valgus or varus force at the fracture site. The posterior displacement of the distal fragment is then corrected by applying a force to the posterior aspect over olecranon process and distal part of humerus while the elbow is gently hyperflexed and the elbow is secured in hyperflexion by strapping arm with forearm. The elbow is placed in the lateral position directly on the image intensification. The fracture is fixed with 1.2mm to 2.0 mm K-wires depending upon the age of the patients. In the coronal plane the pins are placed with an angle of 30° with the long axis of the humerus. Elbow was immobilized in 900 flexion.
10 patients were treated with closed reduction and strapping of elbow around chest. The closed reduction was obtained in similar manner described above.
Following achieving the reduction an above elbow POP slab is applied with the elbow at 110° flexion. This is then followed by strapping the elbow around the chest with dynaplast. The idea of strapping is to prevent the rotation at the fracture site by preventing movements at shoulder and elbow.
The patients were called for follow up after 3 weeks and the POP slab was removed. Active range of motion exercises was encouraged. A special mention and warning was given after the removal of splint about avoiding massage and passive stretching of elbow joint. Further follow up were done at 12 weeks and 24 weeks. The patients were examined clinically and radiologically, assessed for range of motion and carrying angle.

Results

The final results of present study of 37 patients, 30 (81.08%) patients had TYPE III fracture, 5 (13.51%) had TYPE II fracture & 2(5.4%) had TYPE I fracture. Out of 30 patients with TYPE III fracture , 20
( 66.66%) undergone surgical fixation with k wires with 16 patients treated with percutaneous k wire fixation & 4 patients with open reduction & internal fixation with k wires, 13 (65%) patients had excellent results, 6 (30%) patients had good results & 1(5%) patient had fair result. The remaining 10 (33.33%) patients with type III fracture was treated conservatively with closed manipulation & reduction & above elbow pop slab (with elbow in 1100-1200 of flexion) and strapping of elbow around chest. , out of which 6 (60%) patient had excellent results, 2 (20%) patients had good results, 1 (10%) patient had fair result & 1(10%) patient had poor results. Out of 5 patients with type II fracture, 4 (80%) patients were treated conservatively with closed reduction and above elbow pop slab & all had excellent results, 1(20%) patient was treated with open reduction & internal fixation with k wires had good result. Out of 2 patients with TYPE I fracture all were treated conservatively in an above elbow pop slab & all had excellent results according to Flynn’s criteria.
In our study of 37 patients majority of the patients were treated within 24 hours. We have started flexion and extension elbow exercises at the end of 4 weeks and K-wire were removed at 4 weeks and all patients showed radiological union at 4 weeks of follow up.

Discussion

Surgical fixation with closed reduction & percutaneous pinning gives more stable fixation, better anatomical reduction with minimal complication. So it is safe and effective method of fixation. It gives excellent functional and cosmetic results when done at appropriate time for displaced supracondylar fracture of humerus in children.
Moreover the results of closed reduction with POP slab followed by strapping of the elbow around chest in young kids are good when done early after injury. In this method the elbow is flexed at around 110 to 120 degrees which will be a problem if there is gross swelling at the fracture site. Hence one of the requisite for this method of treatment is absence of gross swelling. All the cases operated by this method in this series were operated within 6 hrs of hospital admission before the gross swelling appeared. The cases were observed for signs of compartment syndrome and vascular compromise. In our study no patient had any complications of compartment syndrome or vascular compromise due to this method of treatment. The idea behind strapping of the elbow around the chest is to prevent movements of the humerus preventing the fracture from getting displaced.
Conclusion: The results of surgical fixation with either closed reduction & percutaneous pinning or closed reduction and strapping of elbow around chest were comparable with one another.
Cubitus varus deformity is less with k wire fixation and with closed reduction and strapping of elbow around chest due to better anatomical reduction and prevention of movement at fracture.
Elbow stiffness is less with k wire fixation and with closed reduction and strapping of elbow around chest due to early mobilization of elbow.
The results of Conservative treatment with closed manipulation & reduction with above elbow pop slab (elbow in 1100-1200 flexion) application and strapping of elbow around chest in type III fracture supracondylar fractures of humerus in children are almost similar to the operative group but there is high chance of increased rate of complications like loss of reduction, malunion & restriction of movement especially if it’s done in older age group children.

References

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How to Cite this Article: Vibhute N D, Thomas R, Kavalakkatt T. “A Clinical Study of Functional Outcome After Management of Supracondylar Fracture Humerus in A Child”. Journal Medical Thesis 2017 Jan-Apr ; 5(1):27-31.

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