Correlation of Ama status with specific Biomarkers in Patients of Sandhigata Vata (Osteo Arthritis)
- Conditions
- Primary generalized (osteo)arthritis. Ayurveda Condition: SANDHIGATAVATAH, (2) ICD-10 Condition: M150||Primary generalized (osteo)arthritis. Ayurveda Condition: SANDHIGATAVATAH, (3) ICD-10 Condition: M150||Primary generalized (osteo)arthritis. Ayurveda Condition: SANDHIGATAVATAH,
- Registration Number
- CTRI/2022/11/047327
- Lead Sponsor
- All India Institute of Ayurveda
- Brief Summary
**Introduction:**
**Rationale of study-**
*Agni mandya* or *mandagni* is primarily responsible for pathogenesis of all the diseases in Ayurveda**1**. *Ama,* as a result, which originates due to *mandagni* leads to the causation of different diseases through various pathogenesis**2**. *Ajirna* is such a condition produced due to Agni Dusti with the coexistence of *Ama*. *Ama* generated due to *ajirna*, as and when it is afflicted with predominance of *vata,* produces various *vatavyadhis***3**. Among the various *Vata yadhis* explained in the compendium, *Sandhigata vata* is one such condition which is predominantly affecting the senior citizens of both the sexes along with higher middle-aged group people**4**.
Excessive consumption of fat, high carbohydrate and animal proteins are responsible for the production of oxidative stress through the process of glycation. This process of glycation is intermediated through the formation of **Amadori products5** which later gets converted into **AGEs**. **Advanced Glycation End products (AGEs)** are harmful products which are made-up of combination of proteins or fat with sugars in the blood stream**6**. It can also be formed from the foods which are made with the help of high temperature such as grilling, frying, roasting, toasting or uncooked animal protein base foods which can be explained in terms of *Viruddha ahara**7***, ultimately leads to the development of *ajirna*. High levels of AGEs are responsible for the production of oxidative stress and inflammation at the cellular level. Oxidative stress in the cells of the body may increase the risk of cellular degeneration5.
Non-enzymatic glycation of proteins, such as collagen, results in the formation of Advanced Glycation End products (AGEs). Advanced glycation end products result in pathologic stiffening of cartilage and extracellular matrix and accumulate with age. Advanced glycation end product modification of normal articular cartilage increases its stiffness, increases chondrocyte-mediated proteoglycan degradation, reduces its susceptibility to matrix metalloproteinase-mediated degradation, and decreases proteoglycan synthesis by chondrocytes. These observations parallel findings in osteoarthritic cartilage, which suggests that AGE modification could contribute to the pathogenesis of OA**8**.
The oxidative stress is brought about by Lipid peroxidation by the synthesis of free radical compounds like Malondialdehyde (MDA)**9**. Hence this degenerative process may also incorporate into the bones, joints and marrow which can be implicated as *asthi-vaha srota dusti*. This may act as the principal factor in the genesis of *Sandhigata vata* (Osteo arthritis) from a condition of *Ama.*
The antioxidant defence system which protects the biomolecules against potentially damaging effects of free radicals include antioxidants and antioxidant enzymes e.g. superoxide dismutase (SOD)**10**. These enzymes are gradually denatured by the accumulation of AGEs and malondialdehyde at the cellular level. The imbalance between pro-oxidants and antioxidants gives rise to cellular oxidative stress, which plays an important role in the progression of OA. The Culprit effect of free radical on phospholipid or polyunsaturated fatty acid of membrane of cellular or subcellular organelle is characterized by the process known as lipid peroxidation. Consequently, there is a generation of complex mixture of aldehydes, ketones and polymerization products which react and destroy the biomolecules, enzymes and nucleic acid leading to disease process. Malondialdehyde (MDA) is the most abundant and excellent marker of lipid peroxidation among reactive aldehydes and plays a crucial role in rheumatic diseases**11**. In a study by Munesh kumar and Pravesh Kumar with the title “Concept of Ama dosha with special respect to free radicals†they have presumably concluded that Ama can be corelated with free radicals which are intermediate product of faulty metabolism accumulated in the body and circulation which is causing an obstruction in the srotas (channels). This was directly compared with the accumulation of lipofuscin, amyloid body advanced glycation end product (AGE) and modified protein**12**.There are studies available which explains the quantitative and comparative assessment of lipid peroxidation in patients of OA and RA, but none which establishes a connection between the status of Ama and quantified AGE and lipid peroxidation. There is no study available which assesses the status of Ama in patients of osteo arthritis and its corelation with the status of Advanced glycation end product and lipid peroxidation. Hence this study is taken up to throw ample light on this hidden perspective.
**Relevant Epidemiological data:-**
*Sandhi gata vata* has been widely correlated with the condition of osteoarthritis. osteoarthritis is the second most common rheumatologic problem and it is the most frequent joint disease with a prevalence of 22% to 39% in India**13**. Another epidemiological study reveals the prevalence of osteoarthritis to be 28.7 percent**14**.
**Objectives Of the Research: -**
**Primary Objective-** To Assess the status of Ama in Patients of *Sandhigata vata* with the help of a validated Ama scale along with Evaluation of **Advanced glycation end products (AGEs)** through ELISA-quantitative.
**Secondary Objective****-**
Toestimate the status of Serum **Lipid peroxidation through Malondialdehyde and Super Oxide Dismutase** estimation in context to Ama in patients of *Sandhigata vata*.
To corelate the values of Advanced Glycation End Product and Lipid Peroxidation through Malondialdehyde and Super Oxide Dismutase estimation in patients of *Sandhigata vata*.
**Literature review:**
**Classical-**
The Classical concept of Agni is an important contribution of the Ayurvedic Compendium. The hypofunction of Agni leads to the formation of various diseases**15**. According to Acharya Charaka this hypo-functioning of Agni which when is subjected to various nidana like *abhojana* (excess fasting), *atibhojana* (excess eating) and *vishamasana* (improper diet at improper time) etc, these factors cause the ingested food to become *shuktatva* or *Amlatam* or having Sour entity**15**. This *shuktatva Paka* of *ahara* is undigested food particles which paves the way for *ajirna* which is clinically characterized by *vishambha* (obstruction), *Sadan* (body pain), *shira sula* (headache) *bhrama* (Vertigo) etc which are also the characteristic features of *agni dusti*. This faulty digestion process leads to formation of Ama**16**. This produced *ama* due to *agni dusti* produces the symptoms of *Srota Rodha* (Obstruction of the Srotas), *bala bhramsa* (decrease of strength), *gaurav* (feeling of heaviness), *anila moodhta* (Haphazard /disturbed movement of vata), *alasya* (feeling of laziness), *apakti* (Indigestion), *nishthiva* (salivation/secretion in oral cavity), *mala sanga* (obstruction of mutra/purisha), *klama* (exhaustion)**17**.
**Methodology:**
The present study will be taken up as an Analytical, case control study.
**Type of study design-** The present study is analytical, prospective case control study in which patients of *Sandhigata vata* will be selected through purposive sampling from the OPD/IPD of All India Institute of Ayurveda depending on the clinical features of *Sandhigata vata*. The patients selected on the clinical features of *Sandhigata vata*, in them digital X-ray (Antero Posterior View and Lateral View) of the affected joint will be carried out to assess the classical features of osteo arthritis (Sandhigata Vata). After the diagnosis of *Sandhigata vata* (osteo arthritis), the patients will be assessed on the basis of WOMAC Scale of Osteo Arthritis for disease activity assessment. These patients will be taken up as test group. In these patients, assessment of *ama* will be done with the help of validated scale on *ama* assessment and following which they will be subjected to biochemical investigations of Advanced Glycation End Product though ELISA and Lipid Peroxidation through Malondialdehyde and Superoxide Dismutase Colorimetric estimation. The patients of *Sandhigata vata* who also showing the symptoms of *ajirna* may also be included in the study.
Healthy individuals with no history of any disease for the past 6 months will be evaluated with proper history taking and physical examination as required to ascertain the health status. The laboratory investigations will be done as required to rule out any disease condition. The patients without any systemic, autoimmune, metabolic, infective disease or joint diseaseat the time of inclusion will be taken as control group. The same investigations of test group will be done in control group as well.
The results will be interpreted to assess the status of *ama* in terms of Blood parameters in the test group and control group. The obtained data will be interpreted and computed statistically.
**Study setting and time line:** The study setting will be done in OPD and IPD of All India Institute of Ayurveda. The patient of *Sandhigata vata* will be included after verification of the subjective criteria followed by the objective criteria. During selection of the patient inclusion and exclusion criteria will be followed strictly.
For the Subjects in control group, they will be questioned and assessed regarding history of any systemic, autoimmune, metabolic, infective or joint disorders.
**Time line:** The entire study will be completed in 24 months duration.
**Place of study:** At All India Institute of Ayurveda, New Delhi
**Sample size-** The analytical study will be carried out in at least 162 patients of *Sandhigata vata*.
**N1= m’** **=****(Zα****√(r+1)PQ + Zβ√rP1Q1 + P2Q2)2**
**\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_**
**rδ2**
**n2= rm’ δ= (P1-P2)2 N= n1+n2 α= 0.05 Power- 80%**
**P= P1+ P2 (Q= 1-P)**
**2**
Hence with the above formula the calculated values of n1 = 54 n2 = 162.
The study aims to find out the presence of *ama* in patients of *Sandhigata vata* and wish to compare with those of healthy individuals who are not suffering from any disease. It is assumed that those who do not have *Sandhigata vata* or are healthy may have small presence of *ama* and thus assumed as 5%. Whereas in patients *Sandhigata vata* it is assumed at 20%. It is also assumed that the allocation ratio would be 1:3. Therefore the calculated sample size comes out to be 54 for control group and 162 for the case group.
For Control Group of Healthy individuals 54 patients will be incorporated in the study.
Hence the total sample size will be 216.
**Criteria for selection and method of Data Collection**: The patients will be randomly selected on the basis of the subjective criteria, which are the classical features of *Sandhigata vata*. This will be followed by subjecting the patients to biochemical and radiological investigations which will be accounted as objective parameters. The case report form will be filled up, informed consent will be taken in both the groups and the baseline parameters will be recorded.
**Schedule of Data Collection**:
**Visit 1-**Screening on the basis of classical features of *Sandhigata vata* followed by X-Ray of the affected joint. In the diagnosed cases of *Sandhigata vata* (Osteo arthritis) Assessment of Ama will be done following a validated *ama* assessment proforma developed in the department of Roga Nidana Avam Vikriti Vigyana at All India Institute of Ayurveda, New Delhi. Enrolment of the selected patients followed by subjecting the patients for Blood Collection in the institute laboratory to estimate **AGE and Lipid Peroxidation**.
Patient will be well informed, explained regarding the purpose of the study with help of patient information sheet and written consent will be taken. Only after the written consent patient will be subjected to blood investigations.
The subjects taken as control will be assessed by *ama* assessment proforma after examination of their health status. They can be considered as healthy or disease free.
The subjects of control group will be entitled for same blood investigations of AGE and lipid peroxidation after written consent from the subjects.
**Inclusion criteria:**
**Inclusion criteria for group A (Test Group):-**
1) Adult subjects of either sex between the age group of 30 to 70 years irrespective of chronicity.
2) Presence of cardinal sign and symptoms of *Sandhigata vata.*
3) Digital x-ray depicting the features of osteoarthritis.
4) Patients willing to incorporate themselves in the study.
**Exclusion criteria for group A (Test Group)-**
1) Severe cases of osteoarthritis with stiffness and immobility.
2) Any other conditions of the joints and bones like Rheumatoid arthritis and Ankylosing Spondylitis, Tubercular arthritis or infective Arthritis will be excluded.
3) Any malignant condition of the body.
4) Systemic diseases like Diabetes mellitus, Hypothyroidism etc.
5) Pregnancy
**Inclusion Criteria for Group B (Control Group) –**
1) Healthy individuals within the age group of 30-70 years.
2) Individuals without the history of any systemic disease like hypertension, diabetes mellitus, Hypo/Hyper thyroidism, etc.
3) Healthy individuals who has not suffered from any infective, autoimuune, systemic disease for atleast last 6 months.
**Criteria of Diagnosis:-**
**Subjective criteria of *Sandhigata vata*19**
Ø Sandhi shula
Ø Sandhi shopha
Ø Sandhi Sphutan
Ø Prasaran akunchan a pravritti.
Ø Vata Purna Driti Sparsha
**Objective criteria for Sandhivat20**
Ø features of osteoarthritis in digital x-ray
Ø non-uniform joint space loss
Ø osteophyte formation
Ø cyst formation
Ø subchondral sclerosis
**InvestigationsÂ-**
Laboratory Investigations- (These Tests will be carried out at the time Of First visit) after screening on the basis of diagnostic Criteria. All investigations will be carried out following the Good Laboratory Practices (GLP) in both the Test and Control groups
· Advanced Glycation End products (AGE) Estimation through ELISA- Quantitative method
· Lipid Peroxidation through Quantitative colorimetric analysis by Malondialdehyde estimation and superoxide dismutase estimation
**Radiological Investigations:**
X-Ray Of the affected Joint- AP and lateral View (The joint affected with Sandhi Vata and showing the clinical features will be subjected to X-Ray)
**Estimation of Advanced Glycation End Product** –
Through ELISA Technique with quantitative analysis.
**Estimation Of Lipid Peroxidation21:**
**Reference-** marklund S and marklund G (modified by nandi et al,1998).
The most commonly used method is MDA level measurement through the thiobarbituric acid (TBA) assay. In this assay, MDA forms a 1:2 adduct with TBA producing a colored substance that can be measured by fluorometry or spectrophotometry. Sensitive high-pressure liquid chromatography equipment or spectrofluorometric measurement of iron-based promoters may be utilized for the detection of the low MDA concentrations.
**Principle-** In This method trichloroacetic acid and thiobarbituric acid are added to the serum and the mixture is heated in boiling water bath. The result chromogen is extracted with n-butyl alcohol and the absorbance of the organic phase is determined at the wavelength of 530 nm. The determined values are expressed in terms of Malondialdehyde in nmol/ml.
Reagents required- Trichloroacetic (20 mg%), Sulphuric Acid (0.05 Molar), Sodium Sulphate solution (2 Molar), Thiobarbituric acid (TBA), N-butyl alcohol.
**Calculations-**
**Lipid Peroxidation-= Absorbance Of the test**
**\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ × 6.41 × Volume of solution**
**Volume of sample in test tube**
**Procurement of Necessary ELISA Kits :-**
The ELISA kits for Advanced Glycation End Products, Lipid Peroxidation, and Super Oxide Dismutase will be procured according to the budget estimated subsequently and from reputed, reliable manufacturers like ThermoFisher, Mybiosource depending on the timely availability and cost effectiveness. The related tests or biochemical investigations will be carried out in the Molecular biology laboratory of All India Institute of Ayurveda, New Delhi.
**Parameter for assessment of Study outcomes:-**
Subjective Parameters- WOMAC Scale for Osteo arthritis and validated *ama* assessment scale
Objective Parameters- Values of serum Advanced glycation End product, Malondialdehyde and SOD.
**Study Outcomes-**
**Primary Outcomes**-to assess status of *ama* in *Sandhigata vata* (Osteo Arthritis) along with assessment of Advanced glycation End product and Lipid peroxidation comparison with healthy individuals as control
Study the relationship between the status of Ama, AGE and Lipid Peroxidation in patients of *Sandhigata vata* (Osteo Arthritis)
**Secondary Outcome-** to explore the corelation of Advanced Glycation End product with lipid peroxidation as a marker of oxidative stress in patients of *Sandhigata vata*.
To study the relationship between disease activity score (with WOMAC Score) and Ama in the patients of *Sandhigata vata*.
**Statistical Methods For Analysis :-**
· The variability of the data will be evaluated by mean, standard deviation, and standard error and other suitable methods for analysis.
· The statistical analysis of quantitative data of test group and control group will be compared with the help of Z-test.
· The inter group data of biochemical investigations will be compared through One way ANOVA .
· The status of Ama in both the groups will be compared with Students’ ‘t’ test.
Other statistical methods will be used as required.The statistical Data would be computed through SPSS (Statistical Package for Social Sciences) Software Version 25.0 by IBM and Instat- Graphpad version 3.1.
**Ethical Committee Clearance & No:-**
**Cooperation Required -** The department of Roga Nidana avam Vikriti Vigyana for carrying various permissible tests as required in the study.
- Detailed Description
Not available
Recruitment & Eligibility
- Status
- Not Yet Recruiting
- Sex
- All
- Target Recruitment
- 162
-
- Adult subjects of either sex between the age group of 30 to 70 years irrespective of chronicity.
-
- Presence of cardinal sign and symptoms of Sandhigata vata.
-
- Digital x-ray depicting the features of osteoarthritis.
-
- Patients willing to incorporate themselves in the study.
-
- Severe cases of osteoarthritis with stiffness and immobility.
-
- Any other conditions of the joints and bones like Rheumatoid arthritis and Ankylosing Spondylitis, Tubercular arthritis or infective Arthritis will be excluded.
-
- Any malignant condition of the body.
-
- Systemic diseases like Diabetes mellitus, Hypothyroidism etc.
-
- Pregnancy.
Study & Design
- Study Type
- Observational
- Study Design
- Not specified
- Primary Outcome Measures
Name Time Method To assess status of ama in Sandhigata vata (Osteo Arthritis) along with assessment of Advanced glycation End product and Lipid peroxidation comparison with healthy individuals as control At the time of first visit (Baseline) Study the relationship between the status of Ama, AGE and Lipid Peroxidation in patients of Sandhigata vata (Osteo Arthritis) At the time of first visit (Baseline)
- Secondary Outcome Measures
Name Time Method To explore the corelation of Advanced Glycation End product with lipid peroxidation as a marker of oxidative stress in patients of Sandhigata vata. To study the relationship between disease activity score (with WOMAC Score) and Ama in the patients of Sandhigata vata.
Trial Locations
- Locations (1)
All India Institute Of Ayurveda
🇮🇳South, DELHI, India
All India Institute Of Ayurveda🇮🇳South, DELHI, IndiaDr Anand MorePrincipal investigator9422025732drmoreanand@gmail.com