Study abstract was presented at the 9th National Veterinary Internal Medicine Congress (Antalya, Turkey, 5–8 May 2011) as an oral presentation. The abstract was published in the proceedings
Acute-Phase Proteins, Oxidative Stress and Enzyme Activities of Blood Serum and Peritoneal Fluid in Cattle with Abomasal Displacement
Version of Record online: 1 NOV 2012
Copyright © 2012 by the American College of Veterinary Internal Medicine
Journal of Veterinary Internal Medicine
Volume 26, Issue 6, pages 1470–1475, November/December 2012
How to Cite
Maden, M., Ozturk, A.S., Bulbul, A., Avci, G.E. and Yazar, E. (2012), Acute-Phase Proteins, Oxidative Stress and Enzyme Activities of Blood Serum and Peritoneal Fluid in Cattle with Abomasal Displacement. Journal of Veterinary Internal Medicine, 26: 1470–1475. doi: 10.1111/j.1939-1676.2012.01018.x
- Issue online: 20 NOV 2012
- Version of Record online: 1 NOV 2012
- Manuscript Accepted: 5 SEP 2012
- Manuscript Revised: 1 MAY 2012
- Manuscript Received: 1 DEC 2011
- The Scientific and Technological Research Council of Turkey. Grant Number: 108O828
- SUBAPK. Grant Number: 09401110
- Acute-phase response;
- LDA ;
Blood serum and peritoneal fluid acute-phase proteins, oxidative stress indicators, and some enzymes could be used for evaluation of abomasal tissue damage because of displacement in displaced abomasum (DA) cases.
The aim of this study was to investigate the concentrations of acute-phase proteins, oxidative stress indicators, and activities of enzymes in blood serum and peritoneal fluid in cattle with right displaced abomasum (RDA) and left displaced abomasum (LDA) and in healthy cows.
A total of 60 Holstein Friesian cows in early lactation were used, 31 with left and 9 with right displaced abomasum without volvulus diagnosis and no other postpartum disease, and 20 healthy cows as a control.
Materials and Methods
DA diagnosis in dairy cows consisted of physical examination, laboratory, and specific DA tests. Acute-phase proteins, oxidative stress indicators, and enzyme activities were measured in blood serum and peritoneal fluid.
In the RDA group, serum haptoglobin (HPG), serum amyloid A (SAA), malondialdehyde (MDA), adenosine deaminase (ADA), myeleperoxidase (MPO), aspartate aminotransferase (AST), creatine kinase (CK, creatine kinase–MB (CK-MB), and gamma-glutamyl transferase (GGT) activity increased significantly, and serum HPG, MDA, ADA, and AST concentrations increased significantly in the LDA group (P < .05). Peritoneal fluid HPG, MDA, ADA, MPO, ALP, GGT, and LDH concentrations increased significantly, whereas NO concentrations reduced significantly in the RDA group, and HPG, MDA, ADA, and TP concentrations increased significantly, whereas concentrations of NO reduced significantly in the LDA group (P < .05).
Conclusions and Clinical Importance
There are acute-phase responses, oxidative stress, and abomasal tissue damage because of displacement in DA cases. Especially, HPG, MDA, ADA, and MPO concentrations can provide specific information to help in understanding these changes.
left displaced abomasum
mean corpuscular hemoglobin concentration
mean corpuscular hemoglobin
mean corpuscular volume
partial carbon dioxide pressure
packed cell volume
partial oxygen pressure
red blood cell
right displaced abomasum
red blood cell distribution width
serum amyloid A
white blood cell
Displaced abomasum (DA) is a frequent, economically significant and multifactorial disease of high milk-producing cows and 80–90% of cases occur within 3–4 weeks postpartum. Although diseases such as fatty liver, ketosis, and hypocalcaemia are risk factors for displaced abomasum cases,[2, 3] the cause of right (RDA) or left displaced abomasum (LDA) cases has not been found by etiology or physiopathology studies. Decreased abomasal contractility, atony, dilatation, decreased ruminal volume, hypocalcemia, endotoxemia, alkalemia, hypergastrinemia, and hyperinsulinemia might be predisposing factors for displacement. A considerable proportion of DA cases occur on the left side than on the right side. The prognosis of abomasal volvulus cases because of ischemic necrosis of abomasal wall is poor and the survival rate is 61–74%.[3, 6, 7]
Peritoneal fluid analysis is a useful diagnostic method in abdominal disorders because this fluid generally reflects conditions in the peritoneal cavity. Examination of peritoneal fluid aids in the diagnosis of infectious or chemical peritonitis, intestinal wall infarction, digestive system perforation, bladder rupture, biliary system infiltration, intraperitoneal hemorrhage, and peritoneal neoplasia.[8, 9]
Acute-phase proteins (haptoglobin and serum amyloid A), oxidative stress indicators (malondialdehyde and nitric oxide), proinflammatory cytokines, and some enzymes (myeloperoxidase, lactate dehydrogenase, alkaline phosphatase, creatine kinase) can be used to evaluate inflammatory conditions, ischemic effects, and tissue damage caused by obstruction, strangulation, or both in gastrointestinal disorders.[10-13] We tested the hypothesis that changes in oxidative stress, tissue damage, and inflammation in cattle with displaced abomasum can be determined by evaluation of blood serum and peritoneal fluid parameters.
The aim of this study was to investigate the levels of acute-phase proteins, oxidative stress indicators, and some enzymes in blood serum and peritoneal fluid in RDA and LDA cases and in healthy cows.
Materials and Methods
A total of 60 Holstein Friesian cows at 2–4 weeks postpartum (14–30 DIM) were used in this study; 40 cows (2–6 years old) with displaced abomasum (31 LDA and 9 RDA without volvulus) and no other postpartum disease. As for control group, 20 healthy cows (2–4 years old) were selected from healthy cows in the same farm under the same environmental and management conditions. The control cows were also at 2–4 weeks postpartum (14–29 DIM) and diagnosed to be healthy by analyzing their clinical and hematologic status.
The study was approved by the Ethic Committee of Selcuk University Faculty of Veterinary Medicine. All animals were examined for postpartum diseases (mastitis, metritis, retentio secundinarium, pneumonia, hoof problems, ketosis etc) and positives were excluded.
Blood and Peritoneal Fluid Sampling
Blood was taken by jugular venipuncture. Peritoneal fluid samples from all cows were taken by abdominocentesis. The hair was clipped from umbilical scar region (10 cm caudal to the umbilical scar and 10 cm laterally off the midline toward the right inguinal region). The skin at umbilical scar region was prepared aseptically and was injected subcutaneously with 1 mL of 2% lidocaine. Skin and muscle incision was performed with disposable scalpel blade. Then, a metal tip catheter (10-gauge, 11 cm length) was inserted into the incised site and the catheter was passed through the incision site and a 10-mL sterile syringe was attached to the catheter and gentle aspiration was used to obtain peritoneal fluid. While applying gentle aspiration on the syringe, the catheter was moved gently in its full length and in all directions (dorsally, caudally, or ventrally) within the abdominal cavity to maximize fluid retrieval.
The recovery rate of peritoneal fluid samples was 1–8 mL. After collection, the peritoneal fluid was transferred into a tube and was immediately sent to the laboratory for biochemical analyses.
Diagnosis of Abomasal Displacement
Routine physical examination, laboratory tests (hematological tests and blood gases), and specific DA tests (ping on auscultation and percussion, fluid splashing, liptak test, ultrasonography, and lost percussion area of liver in RDA) were performed for DA diagnosis in dairy cows. Dairy cows with clinical DA diagnosis were sent to the Surgery Clinic of Veterinary Medicine for verification.
EDTA blood hemogram1 and gas parameters of heparinized blood2 were evaluated in DA cases and in control animals. Blood serum and peritoneal fluid (without anticoagulant, 5–10 mL) were analyzed by ELISA tests3 for haptoglobin,4 serum amyloid A,5 malondialdehyde,6 nitric oxide,7 adenosine deaminase8 and myleperoxidase.9 Alkaline phosphatase (ALP), alanine aminotransferase (ALT), creatinine kinase (CK), creatinine kinase-MB (CK-MB), γ-glutamyl transferase (GGT), lactate dehydrogenase (LDH), amylase (AML), and total protein (TP) levels in blood serum and peritoneal fluid were measured by an autoanalyzer.10
Analysis of one-way variance (ANOVA) and the Tukey test were used for data evaluation. The Pearson correlation test was used to evaluate the correlation between the data for the same analyte obtained from blood serum and peritoneal fluid of healthy, RDA, and LDA animals. The level of statistical significance was set at P < .05.
Complete Blood Counts and Venous Blood Gas Findings
White blood cell (WBC) counts, red blood cell (RBC) counts, hemoglobin (HGB), red blood cell distribution width (RDW), mean corpuscular hemoglobin concentration (MCHC), partial carbon dioxide pressure (pCO2), bicarbonate (HCO3), and base excess (BE) levels were increased significantly (P < .05) whereas platelet (PLT) count, partial oxygen pressure (pO2), oxygen saturation (O2sat), potassium (K), and chloride (Cl) concentrations were reduced significantly (P < .05) in the RDA group compared with the control group. HGB, RDW, mean corpuscular hemoglobin (MCH), and MCHC levels were increased significantly (P < .05), whereas the PLT count was reduced significantly (P < .05) in the LDA group compared with the control group (Table 1).
|Parameters||Control (n:20)||RDA (n:9)||LDA (n:31)|
|WBC ×103 mm3||11.5 ± 0.67b||13.4 ± 1.58a||9.27 ± 0.84ab|
|RBC ×106 mm3||6.73 ± 0.13b||7.85 ± 0.32a||7.22 ± 0.17ab|
|PLT ×103 mm3||419 ± 20.9a||279 ± 6.51b||272 ± 14.5b|
|PCV%||31.8 ± 0.65||34.8 ± 1.64||33.1 ± 0.96|
|HGB g/dL||9.39 ± 0.21b||11.2 ± 0.44a||11.3 ± 0.28a|
|RDW%||18.0 ± 0.26b||20.8 ± 0.46a||20.4 ± 0.36a|
|MCV fl||47.4 ± 0.75||44.4 ± 1.67||46.1 ± 1.09|
|MCH pg||13.9 ± 0.27b||14.4 ± 0.42b||15.8 ± 0.32a|
|MCHC g/dL||29.5 ± 0.24c||32.5 ± 0.84b||34.4 ± 0.44a|
|pH||7.44 ± 0.01||7.46 ± 0.01||7.43 ± 0.01|
|pCO2 mmHg||37.8 ± 0.77b||42.9 ± 1.95a||35.8 ± 1.04b|
|pO2 mmHg||39.1 ± 1.95a||31.2 ± 2.94b||33.9 ± 1.34ab|
|O2SAT%||73.4 ± 2.24a||60.5 ± 6.07b||65.4 ± 2.02ab|
|HCO3 mmol/L||25.1 ± 0.37b||30.4 ± 2.18a||23.8 ± 1.09b|
|BE mmol/L||0.99 ± 0.42b||6.66 ± 2.38a||−0.41 ± 1.24b|
|Na mmol/L||141 ± 1.13||139 ± 1.87||143 ± 0.98|
|K mmol/L||3.49 ± 0.09a||2.86 ± 0.23b||3.18 ± 0.08ab|
|Cl mmol/L||109 ± 1.69a||98.3 ± 1.74b||104 ± 1.31a|
Serum and Peritoneal Fluid Biochemical Parameters
In blood serum, HPG, SAA, MDA, ADA, MPO, AST, CK, CK-MB, and GGT concentrations were increased significantly (P < .05) in the RDA group compared with the control group. HPG, MDA, ADA, and AST concentrations were increased significantly (P < .05), whereas ALT and NO concentrations were reduced significantly (P < .05) in the LDA group compared with the control group (Table 2). In the peritoneal fluid, HPG, MDA, ADA, MPO, ALP, GGT, and LDH concentrations were increased significantly (P < .05) compared with the control group, whereas NO levels were reduced significantly (P < .05) in the RDA group compared with the control group. HPG, MDA, ADA, and TP concentrations were increased significantly (P < .05), whereas concentrations of NO were reduced significantly (P < .05) in the LDA group compared with the control group (Table 3).
|Parameters||Control (n:20)||RDA (n:9)||LDA (n:31)|
|HPG ng/mL||39.5 ± 14.4c||1516 ± 105a||981 ± 90.5b|
|SAA ng/mL||23.3 ± 3.73b||66.7 ± 18.2a||41.8 ± 6.11ab|
|MDA mmol/L||4.44 ± 0.16b||7.51 ± 0.72a||6.49 ± 0.28a|
|NO μmol/L||23.4 ± 2.09a||18.6 ± 2.73ab||12.9 ± 0.91b|
|MPO Eu/mL||1.29 ± 0.48b||15.8 ± 5.43a||2.97 ± 1.31b|
|ADA U/L||38.8 ± 4.62b||202 ± 42.8a||229 ± 27.2a|
|ALP IU/L||46.6 ± 2.87ab||60.3 ± 10.9a||39.1 ± 3.77b|
|ALT IU/L||20.0 ± 1.23a||18.1 ± 1.85ab||14.5 ± 1.15b|
|AST IU/L||66.9 ± 1.86b||136 ± 27.0a||123 ± 16.5a|
|AMY IU/L||52.2 ± 8.21||44.0 ± 4.12||35.1 ± 1.77|
|CK IU/L||147 ± 16.7b||575 ± 162a||358 ± 52.5ab|
|CK-MB IU/L||244 ± 20.4b||782 ± 198a||502 ± 65.3b|
|GGT IU/L||21.5 ± 1.51b||82.1 ± 17.1a||34.9 ± 3.79b|
|LDH IU/L||1621 ± 52.7||1597 ± 157||1533 ± 73.1|
|TP g/dL||6.79 ± 0.09||6.51 ± 0.24||6.45 ± 0.08|
|Parameters||Control (n:20)||RDA (n:9)||LDA (n:31)|
|HPG ng/mL||15.2 ± 4.76c||1225 ± 147a||617 ± 72.8b|
|SAA ng/mL||61.1 ± 10.0||109 ± 19.0||107 ± 17.9|
|MDA mmol/L||3.85 ± 0.17c||6.89 ± 0.63a||5.85 ± 0.29b|
|NO μmol/L||60.2 ± 3.97a||27.1 ± 14.1b||13.9 ± 4.05b|
|MPO Eu/mL||1.48 ± 0.82b||15.8 ± 8.63a||1.68 ± 0.82b|
|ADA U/L||23.7 ± 3.76c||162 ± 24.9a||99.6 ± 11.0b|
|ALP IU/L||11.7 ± 2.12b||27.2 ± 6.86a||17.1 ± 3.59ab|
|ALT IU/L||2.65 ± 0.41||4.11 ± 1.43||3.96 ± 0.65|
|AST IU/L||33.4 ± 4.19||52.2 ± 11.2||55.4 ± 6.40|
|AMY IU/L||12.2 ± 1.88||18.6 ± 3.28||14.6 ± 1.38|
|CK IU/L||127 ± 38.8||132 ± 47.8||90.1 ± 19.5|
|CK-MB IU/L||146 ± 40.8||182 ± 57.8||118 ± 20.8|
|GGT IU/L||9.80 ± 1.54b||20.0 ± 3.12a||16.7 ± 2.22ab|
|LDH IU/L||348 ± 59.2b||736 ± 204a||482 ± 87.0ab|
|TP g/dL||1.97 ± 0.16b||2.66 ± 0.28ab||2.87 ± 0.20a|
The MDA, CK, CK-MB, and GGT enzyme activities in the control group, as well as the MDA levels in the LDA group, in the blood serum and in the peritoneal fluid were positively correlated (P < .05) (data not shown).
The results of this study indicated that abomasal tissue damage, acute-phase response, and oxidative stress occurred in cattle with left or right displaced abomasum.
In this study, it was concluded that changes in hematological parameters (thrombocytopenia and high RBC, HGB, RDW, and MCHC levels, Table 1) might be as a result of hemostatic dysfunction or disseminated intravascular coagulopathy (DIC). Hemostatic dysfunction and DIC might be as a result of defects in the abomasal mucosa. Lesions in the major abomasal vessels (hemorrhage and thrombosis in gastric and gastroepiploic vessels) because of abomasal necrosis is dependent on the tightness of the volvulus, the duration of ischemia to the abomasum or both rather than on the presence of a thrombosis or rupture of a major vessel; thus, DIC can be an important risk factor for mortality in DA cases. However, for the determination of hemostatic dysfunction and DIC, some specific parameters such as fibrinogen, anti-thrombin III, activated partial thromboplastin time, and prothrombin time in DA cases should be evaluated. High levels of WBC (Table 1) in RDA cases might be the result of an immunological response to endotoxemia, abomasitis, or peritonitis.
It is known that HGP has antioxidant, anti-inflammatory, immune regulation, and antibacterial properties, and thus can be used to detect the presence of disorder/damage, and can aid in preventing a disorder in determining a more precise prognosis and better treatment. The 3 bovine forestomachs and the abomasum can produce APPs, and SAA could be identified only in the abomasum, and expression of HGP was high in the forestomachs and abomasum. It was reported that concentrations of HPG were increased in cattle with DA, HPG and SAA were increased in cattle with DA owing to fatty liver and were increased in cases of fatty liver, mastitis,[22, 23] and traumatic reticuloperitonitis. In this study, it was observed that an acute-phase reaction could be generated as a result of tissue damage caused by abomasal tension and increased intraluminal pressure. It is concluded that oxidative stress and the acute-phase reaction, as well as inflammatory response and tissue damage, are severe in RDA cases, but moderate in LDA cases when the increase in hematological parameters, APP, MDA, blood serum, and peritoneal fluid biochemical parameters (Tables 1-3) are taken into consideration.
Ischemia/perfusion stimulates the production of free oxygen radicals, and compounds such as NO and MDA are produced. MDA can be used as a sensor of tissue damage and reperfusion, and NO can be used as a sensor of non-reflow phenomenon. In this study, the increased concentrations of MDA and MPO, as indicators of lipid peroxidation and ischemic damage, were concluded to be significant (P < .05) in RDA cases, but not significant (P > .05) in LDA cases (Table 2). Levels of NO in both DA groups were lower than those of the control group (Table 2). It was concluded that oxidative stress and apoptosis can be evaluated by the measurement of MDA and NO levels. SAA and HPG concentrations can also be used for evaluation of an inflammatory response.
Concentrations of extracellular adenosine are increased significantly in ischemia, hypoxia, trauma, stress, convulsions, and inflammatory cases. The ADA activity is an important factor in acute and chronic inflammatory responses, and macrophages are its extracellular source.[27, 28] The ADA activity in serum or pleural fluid is considered a marker of specific diseases and pathological process.[29-31] It was reported that serum ADA activity is increased in some diseases, including trichophytosis (12.7 U/L), anaplasmosis (8.81 U/L), and theileriosis (16.4 U/L). The ADA activity in pleural fluid with concentrations >40 U/L has a value in diagnosis of tuberculosis in human patients. In this study, there was a significant increase (P < .05) in ADA concentration in blood serum and peritoneal fluid in LDA cases (blood serum 229 U/L and pleural fluid 99.6 U/L) and in RDA cases (blood serum 202 U/L and peritoneal fluid 162 U/L) as compared with those of the control group (blood serum 38.8 U/L and peritoneal fluid: 23.7 U/L) (Tables 2 and 3). Increased ADA activity might be related to abomasal tissue inflammation and damage caused by the displaced abomasum. High levels of ADA activity in blood and peritoneal fluid in RDA cases compared with LDA cases may be a useful parameter to evaluate the progression of abomasal damage.
It was reported that the activity of MPO in plasma and peritoneal fluid was increased significantly during pathological displacement of intestines and inflammation in horses. Concentrations of MDA and MPO were increased and concentrations of NO were reduced significantly as a result of tissue damage caused by hypoxia during experimental cases of ischemia-reperfusion damage of the mesenterium of rats. Significantly increased concentrations of MDA and MPO are an indicator of oxidative stress and inflammatory response in relevant tissues during experimental abdominal compartment syndrome in rats. Adenosine release is stimulated by hypoxemic intestinal tissues, and there is a strong correlation between hypoxia and splanchnic ischemia. In this research, severe inflammation, tissue necrosis, and neutrophil response[34, 35, 37] were observed in abomasal tissue during RDA when the significant increase (P < .05) in MPO concentration and WBC in RDA cases was taken into consideration. The nonsignificant MPO increase in LDA cases can be explained because this parameter might be an indicator related to changes in the severity of inflammation, neutrophil response, and tissue necrosis.
An increase in serum CK and CK–MB activity is generally considered related to damage of skeletal muscles or the myocardium and possible changes in activity of this enzyme were reported for colon problems and neoplasia cases. The greater increment in serum CK activity and more severe clinical signs and abomasal damage in RDA cases compared with LDA cases suggest that this increase might be as a result of abomasal damage. Therefore, further research is needed to investigate the relationship between CK and CK-MB activity and myocardial ischemia in DA.
Analysis of peritoneal fluid provides useful information for determination of abdominal disorders, propagation, and reasons for inflammation and/or damage.[8, 9] LDH activity in the peritoneal fluid might be increased in liver cirrhosis, bacterial peritonitis, and peritoneal tuberculosis. Amylase activity might be increased in pancreatitis, pancreas tumor, and intestinal perforation. ADA activity might be increased in peritoneal tuberculosis and liver cirrhosis. ALP activity and GGT activity might be increased in obstruction, strangulation, intestinal perforation, traumatic hemoperitoneum, hepatitis, and peritoneal carcinoma. In this research, there were significantly increased concentrations of MDA and HPG (P < .05) and significantly decreased (P < .05) concentrations of NO in LDA and RDA cases. Significantly increased (P < .05) ADA activity during LDA and significantly increased (P < .05) ADA, MPO, ALP, GGT, and LDH during RDA indicate oxidative/inflammatory changes in the abdomen.[24, 35, 36, 42] The concentration of MDA can be used in the evaluation of oxidative changes in the peritoneal fluid.[43, 44] The ADA activity[26, 30, 36] and the concentration of HPG[24, 42] in the peritoneal fluid can be used in the determination of inflammatory response,[34, 37, 45] whereas ALP, GGT, and LDH enzyme activity can be used in demonstrating tissue damage.[35, 41] In consideration of the correlation between blood serum and peritoneal fluid parameters, only LDA cases in the DA group had a positive correlation (P < .05), which suggests that, generally, there is no correlation between peritoneal fluid and blood serum parameters in DA cases.
It is concluded that there are acute-phase response, oxidative stress, and abomasal tissue damage because of displacement (obstruction and increased luminal pressure) in DA cases, and evaluation of serum concentrations of HPG, SAA, MDA, NO, ADA, MPO, AST, CK, CK-MB, and GGT, and peritoneal fluid concentrations of HPG, MDA, NO, ADA, MPO, ALP, GGT, and LDH can provide information to help in understanding these changes. Although these results are promising, clinical and prognostic uses of these parameters warrant further research.
This study was supported by The Scientific and Technological Research Council of Turkey (TUBITAK, 108O828) and SUBAPK (BAP, 09401110).
Conflict of Interest: Authors disclose no conflict of interest.
Vet MS4-e, Suisse, Switzerland
GEM-Premier 3000, MA
MWGt Lambda Scan 200
HPG, ICL Bovine Haptoglobin Kit, E-10HPT, ICL, Inc, OR
SAA kit, USCN Life Science, E90885Bo, Seattle, WA
MDA, Bioxytech-MDA 586, Oxisresearch, Portland
NO, Colorimetric Assay Kit, 780001 Cayman Chemical Co, MI
ADA, ADA Kit, Biosupply, UK
MPO kit, IMMCO Diagnostics, NY
ILab 300 Plus, Instrumention Laboratory Company, Milan, Italy
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