Indian Journal of Animal Research

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Research Article

Indian Journal of Animal Research, volume 55 issue 8 (august 2021) : 910-916

Studies on Biomarkers of Hepatic Lipidosis in Transition Cows with special reference to Liver Ultrasonograhy, Liver Specific Enzymes and Acute Phase Proteins

 

Randhir Singh1,*, S.N.S. Randhawa2, C.S. Randhawa1, Sushma Chhabra1, Naimi Chand3
1Department of Veterinary Medicine, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141 004, Punjab, India.
2Department of Microbiology, Khalsa College of Veterinary Sciences, Amritsar-143 001, Punjab, India.
3Division of Cattle Physiology and Reproduction, ICAR-CIRC, Meerut-250 002, Uttar Pradesh, India.
Cite article:- Singh Randhir, Randhawa S.N.S., Randhawa C.S., Chhabra Sushma, Chand Naimi (2021). Studies on Biomarkers of Hepatic Lipidosis in Transition Cows with special reference to Liver Ultrasonograhy, Liver Specific Enzymes and Acute Phase Proteins . Indian Journal of Animal Research. 55(8): 910-916. doi: 10.18805/ijar.B-4136.

ABSTRACT

Background: Use of novel biomarkers is the need of hour for prediction and early diagnosis of bovine production diseases such as hepatic lipidosis (HL). Liver ultrasonography and estimation of liver specific enzymes activities have been successfully used for diagnosis of HL. However, use of inflammatory mediator like tumor necrosis factor-á (TNF–á) or acute phase proteins (APPs) like C reactive protein (CRP), serum amyloid A (SAA) and haptoglobin (Hp) as biomarkers of HL has not been elucidated. The present study was therefore designed to evaluate liver ultrasonography and serum APPs concentrations as early predictive biomarkers of bovine HL.

Methods: This study included one hundred one (101) multiparous crossbred cows in advanced pregnancy from dairy farm in Punjab, India. The cows were grouped according to their transition stage i.e., Far off dry (FOD), close up dry (CUD) and Fresh (F). Liver ultrasonography along with hemato-biochemical analysis and estimation of acute phase proteins was carried out during different stages of transition period.

Result: This study included 101 cows, of which 71 (70.3%) had ultrasonographic features of normal liver and 30 (29.7%) had ultrasonographic features of HL (Grade I, 10 cows; Grade II, 12 cows; Grade III, 8 cows). Cows with HL had significantly lower levels of hemoglobin (Hb), packed cell volume (PCV), total erythrocyte count (TEC), total plasma protein (TPP), glucose, total calcium (Ca) and zinc (Zn) and significantly higher levels of plasma urea nitrogen (PUN), non-esterified fatty acid (NEFA), beta hydroxyl butyric acid (BHBA), alkaline phosphatase (ALP), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), glutamate dehydrogenase (GDH), lactate dehydrogenase (LDH), TNF–á, CRP, SAA and Hp compared to the negative controls. The APPs, liver ultrasonography, liver enzymes along with NEFA and BHBA could be used as biomarkers for prediction and early diagnosis of HL in cows.

INTRODUCTION

Hepatic lipidosis (HL) is a common metabolic disorder that affects almost half of all the dairy cows immediately post-partum (PP) (Ametaj, 2005), causing major economic losses due to liver-related morbidity and mortality (Bobe et al., 2004). Although much research has been done towards understanding the metabolic events related to development of HL and negative energy balance (NEB) is highly implicated factor, the exact pathological mechanisms still remain unclear. HL results from excess hepatocyte triglyceride (TAG) accumulation due to NEB in high producing dairy cows (Murondot et al., 2004). This NEB leads to non-esterified fatty acids (NEFA) mobilization from adipose tissue along with increased β-hydroxybutyrate (BHBA), thus initiating a cascade of events like increased uptake of NEFA and TAG synthesis by hepatocytes (Melendez et al., 2009). Therefore, serum NEFA and BHBA concentrations are commonly used biomarkers of NEB and fat mobilization in dairy cows. HL predisposes dairy cow PP to several other metabolic disorders, such as clinical and subclinical ketosis (SCK), metritis, laminitis and mastitis. Further, it is also responsible for delayed response to any treatment due to derailed metabolism, thus negatively affecting the production and reproduction status of cows.
 
The severity of HL can be evaluated by liver USG, along with estimation of several laboratory tests (Tharwat, 2012; and Singh et al., 2018). Additionally, estimation of various inflammatory mediators and acute phase proteins (APPs)  like tumor necrosis factor-α (TNF-α), serum amyloid A (SAA), C-reactive protein (CRP) and haptoglobin might lead to better understanding of prediction and prognosis of HL. The acute phase proteins are liver-produced plasma proteins, and their plasma concentration change in response to changes in pro-inflammatory cytokine concentrations in face of systemic inflammation. These changes are part of the acute phase response (APR) (El-Deeb and El-Bahr, 2017 and Mutara, 2007). Being target sites for APPs synthesis, any injury to hepatocytes or change in liver parenchyma (fat infiltration due to NEB) can directly affect the concentration of APPs in the body (Montagner et al., 2016). Although, studies have been undertaken in dairy cows to elucidate the changes in concentrations of APPs in few disorders such as mastitis, parasitic and bacterial infections (El-Deeb and El-Bahr, 2017 and Thomas et al., 2018), however there are limited studies on evaluation of APPs during the transition period and assessing their utility as an early diagnostic and predictive biomarkers of production diseases like SCK and HL in dairy cows (El-Deeb and El-Bahr, 2017; Bossaert et al., 2012 and Komeilian et al., 2011) />        
Evaluation of biomarkers of HL in prepartum dairy cows is of utmost importance to prevent development of HL after parturition. The present study was therefore designed to evaluate liver ultrasonography and serum APPs concentra tions as early predictive biomarkers of bovine HL.

MATERIALS AND METHODS

Animals
 
This study included one hundred one (101) multiparous crossbred cows in advanced pregnancy from dairy farm in Punjab, India. The cows were grouped according to their transition stage as following:
Far off dry (FOD): >10 days after dry-off and not <30 days’ pre-partum.
Close up dry (CUD): expected to calve within 3 to 21 days.
Fresh (F): > 3days’ post-partum to 30 days in milk (DIM).
 
Liver ultrasonography for diagnosis of HL
 
All the cows were examined at each transition stage for HL by using portable ultrasound machine (Sonosite M-turbo; 2-5 MHz convex transducer). Ultrasonography was performed in B-mode. The liver was examined starting from caudal (12th ICS) to cranial (7th ICS) and from dorsal to ventral of each ICS (Tharwat, 2012). Occasionally, color Doppler was used in some moderate to severe cases of HL where identification of the hepatic vessels was difficult. The final grading of HL was done as described by Singh et al., (2018) and Komeilian et al., (2011) i.e., Normal liver, regular homogenous granular parenchymal echo texture, along with obvious sharp margins and no vessel blurring (Fig 1).  Grade I (mild) HL, bright pattern of the liver parenchyma, with slight vessel blurring, but no marked deep attenuation (Fig 2).
 

Fig 1: Ultrasonograms of normal liver of cows; note the homogenous granular echo-texture of parenchyma and sharp obvious margins of vessels. (PV: Portal vein).


 

Fig 2: Ultrasonograms of grade I HL in cows; note the bright pattern, vessel blurring and hyperecogenicity of near field but deep attenuation is not marked. (PV: Portal vein, OMA: Omasum).


 
Grade II (moderate) HL, bright pattern of liver parenchyma, with vessel blurring and appreciable marked deep attenuation (Fig 3). Grade III (severe) HL, bright patter liver parenchyma with complete vessel blurring and marked deep attenuation and no appreciable blood vessels, which were difficult to identify. There is hyper-echogenicity of the near field (Fig 4).
 

Fig 3: Ultrasonograms of grade II HL in cows; note the bright pattern, vessel blurring and presence of obvious deep attenuation. (PV: Portal vein).


 

Fig 4: Ultrasonograms of grade III HL in cows; note the bright pattern, vessel blurring and marked deep attenuation along with hyperecogenicity of near field. (PV: Portal vein).


 
Laboratory test
 
Laboratory testing was done in the cows with HL based on liver ultrasonography. Total 08 apparently healthy cows in which the liver ultrasonographic echo-texture and hepatic vessels were normal, served as controls. The hematological parameters viz. hemoglobin, hematocrit, erythrocyte count and leukocyte count were estimated by using hematology analyzer (ADVIA 2120, Siemens). Biochemical parameters viz. total plasma protein (TPP), albumin, triglycerides, plasma urea nitrogen (PUN), creatinine, glucose, sodium, potassium, aspartate transaminase (AST), alkaline phosphatase (ALP), g-glutamyl transferase (GGT) and lactate dehydrogenase (LDH) were estimated by using biochemistry analyzer (Orthodiagnostic’s Vitros 350 biochemistry analyzer). Glutamic dehydrogenase (GDH) and arginase activities were estimated as per the method of Ellen et al., (1992) and Mia and Koger (1978), respectively. Plasma concentrations of non-esterified fatty acids (NEFA) and β-hydroxybutyric acid (BHBA) were measured by ELISA (Diasys Diagnostics systems kits, Germany).
       
Digested plasma samples were used for measurement of copper, zinc and iron (Fe) using atomic absorption spectrophotometry (Perkin Elmer Analyst 700, USA). Total calcium and magnesium were measured by mixing 0.1 mLof plasma with 9.9 mLof 0.1% lanthanium chloride. Plasma inorganic phosphorus (Pi) was determined using colorimetric method given by Tausky and Shorr (1953).
 
Measurement of acute phase proteins
 
Measurement of serum SAA, CRP, haptoglobin and TNF-α was done in 6 of the 8 control cows, in 5 of 10 cows with mild HL, in 5 of 12 cows with moderate HL and in 4 of 8 cows with severe HL, randomly selected. The serum samples of these same cows were analyzed from three different stages (FOD, CUD and fresh) of transition period. SAA and CRP concentrations were measured by ELISA supplied by the USCN Inc. Haptoglobin and TNF-α concentrations were measured by ELISA (ALPCO, Inc. and MyBioSource, Inc. USA, respectively).
 
Statistical analysis
 
Statistical analyses were done using SPSS (16.0). Analysis of variance (ANOVA) followed by post-hoc pairwise Duncan’s multiple range tests (DMRT). All tests were 2-tailed and P<0.05 was considered significant.
 
Ethical approval
 
All the procedures have been carried out in accordance with the guidelines laid down by the Institutional Ethics Committee and in accordance with local laws and regulations.

RESULTS AND DISCUSSION

Liver ultrasonography for diagnosis of HL
 
Among 101 cows examined, 71 (70.3%) had normal liver USG features while 30 (29.7%) had USG features of HL, including 10 (33%) with grade I HL, 12 (40%) with grade II HL and 8 (27%) with grade III HL (Fig 1 to 4). Of 101 cows, the portal vein (PV) with its stellate ramification was visible in 61 (60.4%) cases, while the PV was observed without its stellate ramification in 35 (34.7%) cases and was scarcely visible in five (5%). The caudal vena cava (CVC) was detected in only 30 (29.7%) cases (Fig 1 to 4). The PV and hepatic veins can be clearly seen within the normal hepatic parenchyma, and the parenchyma edges are sharp and normally visible (Braun, 2009). In the present study, hyper echoic liver parenchyma in HL cases could be attributed to increased internal echoes intensity (due to fat deposition) where the liver parenchyma appears white on ultrasonograms. There was a reduction in the sharpness and increase in blood vessel blurring, whereas in many instances, small blood vessels could only be poorly imaged or could not be noted at all. This possibly resulted from swelling of the hepatic tissue infiltrated with fat, compressing the small hepatic blood vessels, as also reported previously (Tharwat, 2012; Braun, 2009 and Sakha et al., 2006). As the distance from the abdominal wall increases, fat containing hepatocytes tend to weaken the echoes (acoustic impedance), ultimately leading to hyper echoic near field. As a result of this acoustic impedance, there is hyper-echogenicity of the area near the abdominal wall, whereas areas far from abdominal wall were imaged hypoechoic.
 
Hemato-biochemical analysis
 
The mean hemoglobin concentration, hematocrit was significantly lower, while the WBC count was significantly higher in cows with severe HL compared to the healthy controls (Table 1). The mean TPP and glucose concentra tions were significantly lower, while means plasma urea nitrogen, BHBA and NEFA concentrations were significantly higher (P<0.05) in all the three HL severity groups compared to the healthy controls. The means of ALP, AST, GGT, GDH and LDH were significantly higher (P<0.05) in cows with moderate and severe HL. Triglycerides concentration was significantly decreased (P<0.05), while arginase activity increased significantly (P<0.05) in moderate and severe HL cases as compared to healthy and mild HL groups. Plasma calcium and zinc concentrations were significantly lower (P<0.05) in all three HL groups compared to the healthy controls, whereas, plasma iron concentration was significantly increased (P<0.05) in the grade I HL group compared to the control, grade II and grade III HL groups. Leukocytosis was observed in all three HL groups as compared to control and may be attributed to intravascular hemolysis, oxidative stress and inflammatory cascade. In the present study, HL was associated with elevated NEFA and BHBA concentrations, both of which are cytotoxic at elevated levels. During NEB phase, key hormone expression and responsiveness to tissue is altered thereby decreasing lipogenesis and increasing lipolysis. Elevated NEFA concentration increases lipogenesis and ketogenesis in hepatocytes whereas, high BHBA concentrations decreases gluconeogenesis (Wathes et al., 2009 and Braun et al., 2005). Hepatocellular enzymes like AST and GDH are useful in monitoring the HL that commonly occurs around parturition. Being more liver-specific enzyme, elevated serum GDH activity is indicative of either sub-lethal hepatocyte injury or hepatocyte death (Singh et al., 2018).
 

Table 1: Hemato-biochemical parameters and Plasma mineral concentrations in healthy cows (control) and cows with mild, moderate and severe HL.


 
Previously, elevated GDH and GGT concentrations were reported in dairy cows with liver tumors (Kalaitzakis et al., 2007 and Du X et al., 2017) whereas, elevated AST, SDH and GDH activity was documented in fatty liver syndrome cases because of hepatocyte damage (Ok et al., 2013). Hepatic evaluation in dairy animals has also been carried out based on the activities of hepato-cellular leakage enzymes like AST and cholestatic enzymes like GGT (Singh et al., 2018). In present study elevated serum AST and GDH activities were noticed in cows with moderate and severe HL as compared to healthy controls. Although elevated GDH levels are documented in acute hepatic damage (within 4 to 24 hours of hepatocyte injury). It may be suggested that elevated GDH levels were also hallmark in cows with HL, which may either develop acutely within hours or may take few weeks. Previously also, a similar finding of elevated GDH level in fatty liver cases was documented by Sevinc et al., (2001).  A significantly lower serum triglyceride concentration was observed in moderate and severe HL cases as compared to healthy controls. Similar findings of low Tg, cholesterol and HDL-cholesterol levels in cows with fatty infiltration of liver were previously reported by several researchers (Ok et al., 2013; Sevinc et al., 2001 and Lippolis, 2008).
 
Acute phase proteins
 
The mean concentrations of the APPs at three different time points during transition in the healthy controls and the three grade groups of HL are presented in (Table 2). A significant increase (P<0.05) in all APP’s was observed in both groups throughout the transition period. The concentrations of all measured APPs were significantly higher in the three HL grade groups compared to the control group. Although the concentrations of all four measured APPs in the healthy control group increased significantly throughout transition period, their concentrations in the HL groups were significantly higher (P<0.05) compared to the former. This coincided with significantly increased concentrations of serum NEFA and BHBA and other serum chemistry changes suggestive of liver damage. During the transition period, the cow’s immune system is significantly compromised owing to interactions between the immune system and cells of reproductive system, which are responsible for pregnancy maintenance (Enrico et al., 2017). Further, NEB and oxidative stress around parturition is manifested by activation of the APR, ultimately leading to increased production of APPs by hepatocytes (El-Deeb and El-Bahr, 2017). Processes occurring around parturition are associated with various changes in glucose and lipid metabolism, which may include increased lipolysis, accelerated NEFA production and decreased cholesterol (Sordillo et al., 2009). Investigations in human medicine showed that altered lipid metabolism, increased concentrations of NEFA in blood serum, and oxidative stress may markedly influence the systemic inflammatory response, and the development of inflammatory-based diseases (Kushibiki et al., 2002). Previously, in one experimental study (Wood et al., 2009) involving TNF α administration to dairy cattle, increased production of APPs with decreased appetite and cachexia and increased release of NEFA from adipose tissue into the plasma was noted. Therefore, higher NEFA concentration in serum can also be attributed to the activation of the immune system. Some researchers (Ametaj, 2005 and Du X et al., 2017) have reported that higher concentrations of Hp and SAA after calving correlate positively with total lipids in the liver and proposed that HL found in cows could be a response to non-specific inflammation associated to parturition.
 

Table 2: Acute phase proteins at different transition stages in healthy cows (control) and cows with mild, moderate and severe HL.

CONCLUSION

The concentration of APPs increases with severity of fatty infiltration in liver and can be used as a promising early predictive biomarkers for HL along with ultrasonography and other hemato-biochemical parameters.

ACKNOWLEDGEMENT

The authors are highly thankful to the Director of Research and the Head, Department of Veterinary Medicine, GADVASU for providing necessary facilities. The authors thank the participating dairy farm owner for his cooperation throughout the study period.

CONFLICT OF INTEREST

The authors declare no conflict.

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