Liver disease

Investigating Liver Disease
Serum (clotted blood)

1. SERUM ENZYMES
   Where cases of liver disease are diagnosed it is worth considering screening liver enzymes in
   herdmates to establish if any others are suffering from subclinical liver disease. In establishing likely
   causes i.e. toxins in a common feed source, it can also be helpful to test horses that are kept on the
   same property but subject to different feeding and management. Viral infections (e.g. Equine
   Hepacivirus and Parvovirus) are also a consideration although it seems they do not tend to caseu
   problems as an outbreak.
   Alkaline Phosphatase (AP)
   Increased serum AP concentration has the strongest association with failure to survive liver disease of
   any enzymes although increased serum AP concentration is neither consistently increased in liver
   disease nor liver specific. In addition to hepatobiliary sources, serum AP is known to be derived from
   bone, intestine, inflammatory cells and placenta and these possible sources should be considered in
   interpretation of increased serum AP concentrations.
   Gamma-Glutamyl Transferase (GGT)
   Although mild to moderate increases in serum GGT (e.g. up to 100 iu/L) are of limited diagnostic or
   prognostic value, it is nevertheless very unusual to find significant hepatopathy in horses in the
   absence of increased serum GGT. Additionally, marked increases in serum GGT concentration (e.g.

400 iu/L) are associated with a poor prognosis. Modest incre ases in serum concentration of GGT
should be interpreted with great caution as examination of liver biopsy specimens in such cases often
fails to reveal significant underlying liver disease. The pancreas, or even kidneys, could potentially be
the source of increased serum GGT in the absence of hepatopathy although renally-derived GGT is
widely accepted to appear in urine and not serum. Increases in serum concentrations of liver-derived
GGT may result from insults too minor to result in detectable histopathology. For example, horses
with intestinal disease and particularly right dorsal displacement of the large colon are often reported
with increased GGT perhaps as a result of direct pressure applied to the liver by the distended and
heavy colon. Furthermore, cases of liver disease are not infrequently seen
where increasing concentrations of serum GGT may be noted despite clinical evidence of
improvement of the hepatopathy, perhaps as a consequence of reparative processes or biliary
hyperplasia leading to increased serum GGT.
Aspartate Aminotransferase (AST)
AST is derived from widespread tissue sources and has low specificity for liver disease although in the
majority of liver disease cases it will be increased. Muscle is the main alternative source (same for
LDH).
Glutamate Dehydrogenase (GLDH)
Although serum GLDH is generally increased in liver disease, it only has moderate specificity probably
due to fairly mild and innocuous hepatic insults resulting in increased serum GLDH concentrations. Its
relatively short serum half-life might suggest an association between GLDH levels and the currently
active degree of hepatic insult. The prognostic usefulness of GLDH is debatable and very high values
may be encountered in horses that recover uneventfully.

2. MARKERS OF HEPATIC FUNCTION
   Serum concentrations of several biochemical substances have been reported to reflect the capability
   of the liver to perform its normal functions. These are primarily endogenous and exogenous
   substances that should either be eliminated or synthesised by the liver. They include various amino
   acids, ammonia (NH3), bile acids, bilirubin (total (Tbil) and direct (dBil)), fibrinogen, globulins, glucose
   and urea.
   Serum globulins
   Hyperglobulinaemia is a common finding in association with hepatic insufficiency, probably result ng
   from systemic immunostimulation by intestinal-derived antigenic material following loss of the
   protective barrier of Küpffer cells in the liver. When increased serum globulins are found in liver
   disease cases, this is a strong indicatIon that there has been a considerable liver insult and the
   magnitude of the increase in serum globulin concentration has prognostic relevance. Serum globulin
   concentrations greater than 45 g/L are concerning and values as high as 60-70 g/L are occasionally
   seen and warrant a guarded prognosis.
   Serum bile acids
   The main limitation of the usefulness of serum bile acids is that liver disease must be quite severe
   before increased concentrations are detected and most liver disease cases will be found to have
   normal serum bile acid concentration at the time of initial presentation. A normal bile acid should not
   be taken as very reassuring as it only indicates that the liver is coping currently. Arguably this is the
   best time to explore the disease further as specific targeted treatment is likely to be more successful
   than when dealing with a failing liver. Anorexia and inappetance can increase serum bile acids as high
   as 20-30 mol/L in the absence of liver disease. Hepatopathy cases with serum bile acid concentrations
   greater than 20 mol/L are less likely to survive than those with lower values and chronic cases with
   bile acid concentrations above 100 mol/L are almost
   invariably fatal. Occasional acute hepatopathy cases are seen with far higher bile acid values but this
   seems less prognostically helpful in acute cases.
   Serum albumin
   Although albumin is synthesised by the liver, it has a long serum half-life hence marked
   hypoalbuminaemia is uncommon in equine liver disease. Serum albumin concentrations below 20 g/L
   are very rarely encountered even in severe hepatopathies.
   Bilirubin
   Failure of the liver to take up, conjugate and excrete bilirubin may lead to increased serum
   concentrations of unconjugated and/or conjugated bilirubin. Anorexia and haemolysis are additional
   causes of unconjugated hyperbilirubinaemia. The majority of equine liver disease cases have normal
   or only moderate increases in serum bilirubin concentration (typically 50-150μmol/L) and the
   unconjugated fraction usually greatly exceeds the conjugated fraction. Cases of liver disease in the
   horse in which serum conjugated bilirubin represents greater than 25% of total bilirubin are likely to
   have obstruction of the biliary tract.

Urea and creatinine
Low serum urea concentrations have been recognised previously in association with liver failure and
have been suggested to indicate reduced hepatic synthesis of urea from ammonia. Although the
majority of equine hepatopathy cases have normal serum urea concentrations, decreased serum urea
is associated with more severe hepatopathies and has prognostic relevance. Creatinine is also
sometimes low in hepatic disease cases for unknown reasons but perhaps due to washout associated
with polydipsia.
Blood clotting times
Hepatic insufficiency is associated with a decrease in the synthesis and function of the majority of
procoagulant, anticoagulant and fibrinolytic proteins in addition to reduced platelet numbers and
function. Despite the complexities of effects on individual proteins, the net effect of hepatic failure on
haemostasis is invariably impairment of coagulation as determined by prolonged APTT and PT,
although clinical evidence of coagulopathy is less commonly seen than is clinicopathological evidence
of coagulopathy in horses with hepatic insufficiency. The incidence of bleeding disorders associated
with performing a liver biopsy is very low and it is not considered necessary to check clotting times
prior to performing the procedure.
Glucose
Despite the central gluconeogenic role of the liver, plasma glucose typically remains within normal
limits in adult horses with hepatopathy. Hypoglycaemia is more common in foals with hepatopathy.
Ammonia
Although plasma ammonia concentration is increased in nearly all cases of hepatic encephalopathy,
the concentration does not necessarily correlate with severity of the disease. This apparent paradox
may be explained by increased permeability of the blood brain barrier to ammonia in cases of hepatic
encephalopathy. Ammonia has to be assayed within minutes and is therefore not offered via our
referral laboratory unless you can arrange immediate delivery of a chilled sample to us.

3. FURTHER INVESTIGATION OF OUTBREAKS
   When a case of liver disease is identified it is usually found that many other herdmates are also
   affected, albeit often subclinically. The finding of multiple affected horses suggests that an infectious,
   toxic or nutritional problem might underly the problem. Attempts to further identify the aetiology
   may take several possible courses including discussion with owners to identify common managmental
   and dietary factors. Further tests to attempt to identify the cause also include:
   Mycotoxins
   The only 2 mycotoxins recognised to cause hepatopathy in horses comprise aflatoxins and fumonisin
   B. These are possible factors in forage-fed horses and the concentration in hay can be determined,
   albeit with the limitation that they are unlikely to be evenly distributed amongst a large store of hay.
   Viruses
   Several viruses have been identified as possible candidates for outbreaks of hepatopathy but the
   major suspects currently comprise equine hepacivirus (a close relative of human hepatitis C virus)
   and also equine parvovirus.

Liver Biopsy
This is a useful means of ruling in or ruling out causes such as pyrrolizidine alkaloid toxicity and also
to find pathology more typical of the mycotoxins or hepatitis viruses mentioned above. Biopsy will
also help determine prognosis and guide the selection of specific therapies based on the exact type
of hepatopathy present.
Performing a liver biopsy

• The subject is sedated
• Biopsy site and depth are chosen based on ultrasound and site is prepared for a sterile procedure
• 5-10 mL local anaesthetic is infiltrated subcutaneously and through the intercostal muscles to the parietal
  peritoneum using a 21 gauge 11⁄2” needle
• A small stab incision is made through the skin using a no.15 (or 11) scalpel blade
• A 14 gauge 16 to 20 cm biopsy needle is advanced perpendicularly to the skin to the predetermined depth and
  the biopsy is collected (angling cranially creates a larger target but makes automatic needles harder to fire)
• A total of 2-3 cm of biopsy specimen is required. The procedure is repeated if a suitable biopsy specimen is
  not obtained (2 or 3 attempts sometimes required)
• Biopsy specimens are placed in 10% neutral buffered formalin for histopathologic examination and/or plain
  sterile containers for bacteriologic culture
• Samples from at least 2 separate sites are preferable
• Topical antiseptic spray is applied to the skin incision
• A single dose of 2 mg/kg phenylbutazone IV is administered
  Interpretation
  A prognostic biopsy scoring system has been developed at Liphook based on scoring 5 histopathologic
  features (Table 1). This results in a prognostic biopsy score between 0 (best prognosis) and 14 (worst
  prognosis). As a general rule horses with biopsy scores of 0-2 will survive, horses with scores above 8
  generally die and those in between merit aggressive treatment.

Absent Mild Moderate Severe
Fibrosis 0 0 2 4
Irreversible cytopathology 0 1 2 2
Inflammatory infiltrate 0 0 1 2
Haemosiderin accumulation 0 0 0 2
Biliary hyperplasia 0 0 2 4

Table 1. Prognostic biopsy scoring system. Each individual parameter is scored and the total
calculated.
FURTHER READING:
Durham, A.E. et al. (2003) An evaluation of diagnostic data in comparison to the results of liver biop
sies in mature horses. Equine Veterinary Journal 35, 554-559.
Durham, A.E. et al. (2003) Development and application of a scoring system for prognostic
evaluation of equine liver biopsies. Equine Veterinary Journal 35, 534-540.
Durham, A.E. et al. (2003) Retrospective analysis of historical, clinical, ultrasonographic, serum
biochemical and haematological data in prognostic evaluation of equine liver disease. Equine
Veterinary Journal 35, 542-54 7.