3. Marco teórico
2.1. El Burnout y la relación con las variables Sociodemográficas
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APPENDIX 2
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14. Pack year(s) smoked: ………
15. Year of Diagnosis of Diabetes: ……….
16. Duration of Diabetes ………..
17. Medication for diabetes (Specify): ……….
18. Hypertension: Yes { } No { }
19. Duration of Hypertension (years): ………
20. Use of anti-lipid drugs: Yes { } No { }
21. Type of antilipid agent (specify): ……….
22. Intermittent Claudication : Present { } Absent
23. Other abnormal sensation: __________________ (specify)
24. Weight (kg): ……….
25. Height (metres): ………..
26. BMI (kg/m2): ……….
27. Waist Circumference(centimeters) ………..
28. Blood pressure (mmHg) ………
29. Palpation of posterior tibial artery: Present { } Absent { } 30. Palpation of dorsalis pedis artery: Present { } Absent { } 31. Ankle Brachial index
Mild 0.70–0.90 Moderate 0.40–0.69 Severe <0.40
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Section C: Investigations.
32. Plasma glucose (mmol/L) (A) FBS…….. (B) 2hPP…….
33. Glycosylated Hb level(%): ………..
34. Fasting Lipid Profile (mmol/L)
A. Total ………
B. HDL ……….
C. LDL ………..
D. Triglycerides ………..
35. White blood cell count:
Total ………
Differentials (%)
Neutrophils: ……….
Basophils: ………
Eosinophils: ………
Monocytes: ……….
Lymphocytes ………..
36. High sensitivity C-Reactive protein level (mg/l): ……….
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APPENDIX 3
ANTHROPOMETRIC MEASUREMENTS WEIGHT
Weight was measured using standard weighing scale (Hanson,
England) to the nearest 0.1 Kilogram(Kg) with subjects in light clothing.
HEIGHT
Height was measured with patients standing erect without any foot wear or head-tie against a graduated height scale (stadiometer) in 0.01 metres (m).
BODY MASS INDEX (BMI)
BMI was calculated from the weight and height using the following formula:
BMI = Weight(kg) / Height2 (kg/m2).49
BMI was used to categorize patient’s weight as follows49: Classification BMI (kg/m2)
Underweight <18.5
Normal 18.5 -24.9
Overweight 25-29.9
Obese ≥ 30.
WAIST CIRCUMFERENCE (WC)
Waist circumference (WC) was measured with a measuring tape to the nearest 0.1cm and used to categorize them as either obese or not.
Waist circumference was measured halfway between iliac crest and
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lowest rib in mid-axillary line. A waist circumference of ≥ 94cm for male and ≥ 80cm for female will taken as obese.52
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APPENDIX 4
ANKLE BRACHIAL INDEX (ABI) MEASUREMENT
The ABI was measured by placing the patient in supine position for 5-10 minutes.22 Systolic blood pressure was measured in both arms. This was done by placing the blood-pressure cuff on the patient’s right or left arm. The brachial pulse was palpated and gel was applied at the site where the pulse was felt. The tip of the Doppler probe was placed at 45- 60 degree angle where the pulse was felt to obtain a clear Doppler signal.
The cuff was rapidly inflated to 20 to 30 mm Hg above the point of cessation of brachial-artery flow, then it was slowly deflated in order to note the systolic value. The gel was wiped from the patient’s skin and the procedure was repeated on the other arm.53
The cuff, (the same used for brachial) was placed just above the ankle on the right or left leg and the dorsalis pedis pulse was palpated. The
Doppler probe was placed on the palpable dorsalis pedis pulse or on the site that produced the best arterial Doppler signal from the dorsalis pedis artery. Once again, the blood-pressure cuff was inflated to 20 to 30 mm Hg above the level at which flow ceases, then the cuff was deflated slowly and the systolic pressure (the pressure at which the first flow from the dorsalis pedis artery is heard) will be noted. The procedure was repeated
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over the posterior tibial artery.
Then the procedure was done for the contralateral leg to obtain the systolic pressure from both the dorsalis pedis and posterior tibial arteries.53
To calculate the ankle–brachial index, the systolic blood pressure in the ankle in each leg was divided by the higher of the systolic blood
pressures in the arm.22,53
The higher of the systolic pressures from the dorsalis pedis or posterior tibial artery was used as the numerator to determine the ankle–brachial index.22,53 The lower of the two ABI values obtained for the right and the left foot was used for analysis. PAD was diagnosed as ABI < 0.9.
The diagnostic criteria for PAD based on the ABI will be interpreted as follows: 22 ABI between 0.91–1.30 was taken as normal, and value of ≤ 0.9 denoted PAD. Those with ABI of > 1.30 were
excluded from the study.
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APPENDIX 5
GLUCOSE OXIDASE METHOD54
The glucose oxidase method of blood glucose estimation is based on the oxidation of glucose present in the plasma by the enzyme glucose oxidase to form glucoronic acid with the liberation of hydrogen peroxide, which is converted to water and oxygen by the enzyme peroxidase. An oxygen acceptor takes up the oxygen and together with the phenol forms a pink coloured chromogen which can be measured at 500nm.
1ml of glucose oxidase solution was added to a test tube containing 10μl of plasma sample from centrifuged blood specimen in the fluoride oxalate bottles and 10μl of standard glucose solution. Tests on the
standard glucose were performed in the duplicate and there was a “blank”
test tube containing only glucose oxidase solution.
Three test tubes containing plasma samples of known glucose concentrations was also included in each assay run. The tubes were incubated for 5
minutes at 370C in a water bath. Thereafter, when the tubes were cooled to room temperature, the optical densities were read off the spectrophotometer at a wavelength of 500nm. The “blank” test tube was used as a reference point against which the optical densities of all other tubes were
determined. The optical density for the plasma glucose was compared with that for the standard glucose solution which has a known glucose
concentration. The mean values from repeated tests on the standard glucose
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preparation were used in the test samples. The level of glucose was estimated based on the following formula:
Glucose concentration = Absorbance of sample / Absorbance of standard X Concentration of standard.
PRECISION OF PLASMA GLUCOSE ASSAY (i) Intra Assay Precision
Glucose Concentration (mmol) n
CV%
5.89
20 2.19%
14.63 20 1.92%
(ii) Inter Assay Precision
Glucose Concentration (mmol) n
CV%
5.89
10 3.14%
14.63 10 2.9%
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APPENDIX 6
MEASUREMENT OF HAEMOGLOBIN A1c55
Haemoglobin Aic (HbAic) was estimated using whole venous blood with the aid of the Bio-Rad in2it System which allows the in vitro quantitative determination of HbAic. It uses the boronate affinity chromatography to separate glycated fraction from the non-glycated fraction. It is a single- wavelength (440nm) photometer designed as a fully automated system.
The test cartridge contains the sample reagent, as well as wash solution and elution buffer. The venous blood was collected using
specially designed blood keys, which was clicked in place in the cartridge, and the cartridge was placed in the analyser. From this point the reaction is automatic.
The sample reagent lyses the red blood cells and after mixing and incubation, the glycated haemoglobin binds to the boronate affinity resin.
The liquid then flows through the central hub of the cartridge. A frit in the hub retains the affinity resin bound with the glycated haemolobin; the non- glycated fraction is collected and measured photometrically. Wash solution is released, flowing through the central hub to wash the affinity resin.
Elution buffer is released, flowing through the central hub to elute the
glycated haemoglobin off the boronate affinity resin; this glycated fraction is then phometrically measured.
The percentage HbAic (%HbAic) is calculated from the absorbances
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using the following algorithm:
% HbAic = M (Aglycated X 100 / Aglycated + A non-glycated ) + C
“M” and “C” are the slope and intercept factors to correct the value for the Diabetes Control and Complication Trial (DCCT) calibration.
The HbAic result is aligned to the DCCT standards.
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APPENDIX 7
HIGH SENSITIVITY C-REACTIVE PROTEIN ENZYME IMMUNOASSAY TEST44
The high sensitivity C-reactive protein ( hsCRP) ELISA is based on the principle of a solid phase enzyme-linked immunosorbent assay. The assay system utilizes a unique monoclonal antibody directed against a distinct
antigenic determinant on the CRP molecule. The mouse monoclonal anti-CRP antibody is used for solid phase immobilization (on the microtiter wells). A goat anti-CRP antibody is in the antibody-enzyme (horseradish peroxidase) conjugate solution.
Reagents and materials
1 Antibody coated wells—Microtitre wells coated with monoclonal anti C-reactive protein.
2 Reference standard sets
3 C-reactive protein sample diluent
4 C-reactive protein enzyme-conjugate reagent containing anti C-reactive protein conjugated to horseradish peroxidase
5 Tetramethylbenzidine (TMB) reagent substrate solution.
6 Stop solution (1 bottle, 11ml/bottle) contains diluted sulphuric acid.
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6 Distilled water
7 Multi-channel pipettes 8 Absorbent paper 9 Microtitre plate reader
Storage condition of the C-reactive protein kit
1 The kit was stored according to the manufacturer’s instruction (at a temperature of 2-80C) before it was used.
2 The microtitre plates were kept in a sealed bag with desiccant to minimize exposure to moisture.
Reagent preparation
1 The reagents were allowed to reach room temperature before being used.
2 5µl of patients serum was diluted with 495µl of sample diluent.
Assay procedure
1 10µL of standard, specimen, and control samples were added into the appropriate wells of the anti CRP antibody pre-coated microtitre plates.
100µl of CRP enzyme conjugate was added into each well, it was
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mixed and covered, and was incubated at room temperature for 45 minutes.
2 The incubation mixture was removed by flicking the content into a waste container, and was then washed five times with distilled water.
3 Residual water droplets were removed by striking the plate onto adsorbent paper.
4 100µL of Tetramethylbenzidine (substrate solution) was added to each well, it was mixed and was incubated at room temperature for another 20minutes. This resulted in the development of a blue colour.
5 100µL of stop solution (hydrochloric acid) was added into each microtitre plate well, the mixture resulted in colour change from blue to yellow.
6 The optical density was read at 450nm using a microtitre plate reader set within 15 minutes.
Calculations of the assay
The mean absorbance value (OD) for each reference standard, specimen, and control were calculated, and a curve was plotted using the mean absorbance (OD) obtained for each reference standard against its concentration in µg/mL on a graph paper with the absorbance on the vertical (y) axis and concentration on the horizontal (x) axis. Using the mean absorbance value for each sample, the
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corresponding concentration of CRP was determined and was multiplied by dilution factor to obtain the value in mg/L.
PRECISION OF SERUM C-REACTIVE PROTEIN
(i) Intra-Assay Precision – was determined by replicate determinations of five different samples in one assay
Serum Sample 1 2 3 4 5 Replicates 22 22 22 22 20 Mean CRP (mg/L) 0.546 0.894 2.021 3.492 17.549 S.D. 0.041 0.037 0.085 0.146 0.397
C.V. (%) 7.5% 4.1% 4.2% 4.1% 2.3%
(ii) Inter-Assay Precision – was determined by replicate measurements of five different serum samples over a series of individually calibrated assays.
Serum Sample 1 2 3 4 5 Replicates 20 20 20 20 20 Mean CRP (mg/L) 0.490 0.890 1.925 3.529 17.435 S.D. 0.020 0.023 0.078 0.114 0.438
C.V. (%) 4.1% 2.5% 4.1% 3.2% 2.5%
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APPENDIX 8