1. Quantitative analysis of Troponin I serum values in patients with acute cholecystitis Quantitative analysis of Troponin I serum values in patients with acute cholecystitis Babic Ž, Bogdanović Z, Dorosulić Z, Basha M, Krznarić Ž, Sjekavica I, Kujundžić M, Tadić M, Banić M, Jagić V, Marušić M Coll Antropol. 2012 Mar;36(1):145-50

2. INTRODUCTION We studied troponin Ic (cTnI) as a new parameter, that until now, when significantly elevated has represented myocardial hypoxia and infarction]. Non-specific elevations of the cTnI (less than 0.05ug/L), is a sign of myocardial hypoxia but not infarction. Myocardial hypoxia can also result from systemic hypoxia caused by a severe form of acute cholecystitis.

3. PATIENTS AND METHODS MATERIALS Collected from the 65 subjects upon their written informed consents, according to the Helsinki Declaration and the Hospital Ethics Committee. In 65 patients with acute cholecystitis we measured the Troponin I (cTnI) level. Diagnose of acute cholecystitis was inclusion criteria. Acute cholecystitis was diagnosed by standard clinical, laboratory and ultrasound based signs and symptoms. We have used Tokyo Guidelines criteria for severity of acute clolecystitis (moderate, severe)

4. PATIENTS AND METHODS Inclusion criteria A) Clinical symptoms – Local signs of inflammation: upper right quadrant (RUQ) pain 1-2 hours after a meal: mass/pain/tenderness Murphy sign propagates towards the scapula and back in association with elevated body temperature B) Systemic signs of inflammation – Fever – Laboratory signs: high leukocyte count (WBC) elevated serum C-reactive protein value

5. PATIENTS AND METHODS C) Immaging finding characteristic of acute inflammation – CT, MR (enlarged, thickened wall, pericholecystitic fluid, linear high density areas in pericholecystitis fat tissue, pericholecystitis high signal) – Abdominal Ulrasound signs of acute cholecystitis: Sonographic Murphy sign Thickhness of the gallbladder wall > 4 mm with signs of dissection (front wall) presence of incarcerated gallstone, debris, and pericholecystic fluid Enlarged gallbladder (>8cm long axis, >4cm short axis) one in A+, one in B+, and C confirms A and B

6. PATIENTS AND METHODS Tokyo Guidelines (Criteria) – parameters that represented the local status of inflammation – systemic response to acute cholecystitis – depending on disease severity like hypoxia and circulation Mild form – Acute cholecystitis in healthy patient with no organ dysfunction – Mild inflammatory changes in the gallbladder – Safe and Low risk of operative procedure

7. PATIENTS AND METHODS Tokyo Guidelines (Criteria) Moderate form: – Accompained by any one of the following conditions – WBC>18000/mm 3 – Palpable tender mass in the RUQ – Duration >72h – Marked local inflammation: biliary peritonitis, pericholecystitic abscess, hepatic abscess, gangrenous cholecystitis, ephysematous cholecystitis – Difficulty to perform operative procedure

8. PATIENTS AND METHODS Tokyo Guidelines (Criteria) Severe form: – Accompained by any one of the following conditions – Cardiovascular disfunction (dobutamine or dopamine >5ug/kg/min) – Neurological disfunction (  level of consciousness) – Respiratory dysfunction (PaO2/FiO3<300) – Renal dysfunction (olyguria, creatinine) – Hepatic dysfunction (PT-INR>1,5) – Hematological dysfunction (Plt>100000/mm3) – Significantly elevated WBC count – The duration of symptoms >72 h, – A palpable abdominal mass in right upper quadrant – High risk of operative procedure

9. PATIENTS AND METHODS Exclusion criteria -Acute coronary incident - Patients with severe acute cholecystitis were tested for positive coronary artery disease by treadmill stress test (1 month later). -Patients with abnormal renal function (findings of serum and urea values) were excluded -Patients with biliary obstruction (US finding, GGT, ALP) -Patients with complications such as acute pancreatitis or ileus. -Upper gastrointestinal endoscopy was done to exclude all other differential diagnoses. Follow up -Up to two months after an episode of severe acute cholecystitis that was conservatively treated and healed, all patients underwent trans- abdominal or laparoscopic surgical cholecystectomy. -Gallbladder tissue sample analysis was done according to standard procedure. All patients with pathological findings that fulfilled inclusion criteria were included in the study, while other causes of gallbladder diseases were excluded (e.g. tumor, etc.).

10. PATIENTS AND METHODS METHODS The Troponin I measurements -The Troponin I serum values were measured by immunoflorometric method (Dimension, Dade Boehringer). We considered Troponin I values up to 0.5 ng/mL while those values between 0.5-1.5 ng/mL were excluded as a possible acute myocardial infarction. Other measurements -Ultrasound system with convex 3.5 MHz probe. -Laboratory parameters (leukocyte, CRP, amylase, bilirubin, AST, ALT, gamma glutamyl transferase, alkaline phosphatase, urea and creatinine) - measured according to standard procedures. Statistics We used software for statistical analyses that included mean, standard deviation, ANOVA, test of homogeneity of variance and multivariate multiple regression test.

11. TABLE 1. THE MEANS AND RANGES OF PARAMETERS ACCORDING TO TOKYO CLASSIFICATION OF DISEASE SEVERITY IN PATIENTS WITH ACUTE CHOLECYSTITIS _____________________________________________________________________________________ Tokyo classification score of acute cholecystitis _____________________________________________________________________________________ Moderate (score=2)Severe(score=3)P Median RangeMedian Range _____________________________________________________________________________________ Age(yrs.)64.6 37.060.7 47.00.539 Sex(m/f)1.77 1.01.7 1.00.199 Leucocytes(x10 9 /L)12.54 12.714.6 13.00.0284 CRP(mg/L) 19.8 37.749.5 * 233.0 0.0002 Troponin I (ug/L) 0.004 0.050.22 * 0.480.0001 US galblder wall thicknes (mm) 5.5 5.06.36 4.00.011 US /pericholecystitis (y/n)0.16 1.00.27 * 1.00.046 CK(U/L )65.8 82.081.1 135.00.478 Bilirubin(  mol/l) 14.4 28.016.6 31.20.131 AST(U/L)21.1 31.026.4 37.00.451 ALT(U/L)21.5 34.023.1 34.00.767 GGT(U/L)33.3 58.045.1 112.00.171 ALP(U/L)71.0 80.067.0 85.00.066 Amylase-s(U/L)69.10 88.063.6 * 122.00.028 Amylase-u(U/L)131.1 200.0143.0 264.00.518___________________ Statistical significance for Troponin I was at * P <0.05 level (Levene test of Homogeeity).

12. TABLE 2. RESULTS OF SERUM TROPONIN I VALUES IN PATIENTS WITH EITHER MODERATE OR SEVERE FORM OF ACUTE CHOLECYSTITIS _________________________________________________________ Moderate Severe Total ____________________________________ (N)(N) (N) _________________________________________________________ Troponin = 0 ng/mL1612 28 Troponin > 0 ng/mL 235 37 _________________________________________________________ 1847 65 _________________________________________________________ Result of  2 -test presents statisticaly significant diference (P<0.005; with  2 =21.26 and df=1) between the group with troponin level 0.00 ng/mL vrs. the group with troponin level higher than 0.00 ng/ml

13. TABLE 3. TROPONIN I IN MULTIPLE REGRESSION ANALYSIS WITH OTHER PARAMETERS ________________________________________________________________________________ US gallblader wallTroponin Thicknes r P rP. _____________________________________________________________________ Age 0.040.77-0.070.60 SEX0.160.21-0.100.44 Leukocyte0.140.26-0.030.79 CRP 0.42 * 0.001 0.100.42 TROPONIN 0.58 * 0.001 // US/ Gallbladder thickness ( > 6 mm) // 0.58 * 0.001 US/pericholecystitis 0.160.213 0.05 0.68 CPK-0.120.33-0.030.81 BIL 0.130.31 0.080.52 AST 0.090.48 0.27 * 0.03 ALT 0.040.76 0.170.18 GGT 0.220.079 0.26 * 0.048 ALP-0.010.96-0.150.23 Amylase (serm.) 0.050.68 0.100.42 Amylase (urine) 0.080.53 0.100.41 ______________________________________________________________________________ Statistical significance was at * P < 0.05 leve (Multiple regresion analysis).

14. TABLE 4. THE DIFFERENCES BETWEEN THE PARAMETERS ACORDONG TO ULTRASOUND MEASURED GALLBLADDER WALL THICKNESS IN PATIENTS WITH ACUTE CHOLECYSTITIS ____________________________________________________________________________________________ Gallbladder wall thickness (mm) ____________________________________________________________________________________________ 6 x  SDx  SD _____________________________________________________________________________________________ Age(yrs.)58  963  130.067 Sex(m/f)1.66  0.41.74  0.40.257 Leucocytes(x10 9 /L)14  4.4514  3.10.115 CRP(mg/L) 22  1148  55 * 0.00028 Troponin I (ug/L) 0.033  0.090.21  0.19 * 0.00001 US /pericholecystitis (y/n)0.17  0.380.27  0.450.046 CK(U/L )80  3876  280.078 Bilirubin(  mol/l) 17  16  70.531 AST(U/L)22  1126  90.445 ALT(U/L)20  1023  90.657 GGT(U/L)35  1844  220.368 ALP(U/L)65  2269  220.678 Amylase-s(U/L)58  2172  380.006 Amylase-u(U/L)134  60142  630.483 ____________________________________________________________________________________________ Statistical significance for Troponin I was at * P <0.0001 level (Analysis of variance).

15. CONCLUSION Taken together, the determination of Troponin I in conjunction with gallbladder wall thickness, other parameters (leukocyte count, C-reactive protein, AST, GGT etc.) and clinical presentation could be a very sensitive, useful and simple tool, easily incorporated into daily practice. Clinically, it is very important to determine the degree of acute and non obstructive cholecystitis (mild, moderate or severe form, with or without complications). This can lead to a more precise determination of disease severity ensuring timely and appropriate therapy resulting in lower costs and fewer complications.

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