Objective—To evaluate the accuracy of digitally scanned rhodanine-stained liver biopsy specimens for determination of hepatic copper concentration and compare results with qualitatively assigned histologic copper scores in dogs.
Sample—353 liver biopsy specimens from dogs.
Procedures—Specimens (n = 139) with quantified copper concentration ranging from 93 to 6,900 μg/g were allocated to group 1 (< 400 μg/g ), group 2 (401 to 1,000 μg/g ), group 3 (1,001 to 2,000 μg/g ), and group 4 (> 2,001 μg/g ); stained with rhodanine; and digitally scanned and analyzed with a proprietary positive pixel algorithm. Measured versus calculated copper concentrations were compared, and limits of agreement determined. Influence of nodular remodeling, fibrosis, or parenchymal loss on copper concentration was determined by digitally analyzing selected regions in 17 specimens. After method validation, 214 additional liver specimens underwent digital scanning for copper concentration determination. All sections (n = 353) were then independently scored by 2 naive evaluators with a qualitative scoring schema. Agreement between assigned scores and between assigned scores and tissue copper concentrations was determined.
Results—Linear regression was used to develop a formula for calculating hepatic copper concentration ≥ 400 μg/g from scanned sections. Copper concentrations in unremodeled specimens were significantly higher than in remodeled specimens. Qualitative scores widely overlapped among quantitative copper concentration groups.
Conclusions and Clinical Relevance—Calculated copper concentrations determined by means of digital scanning of rhodanine-stained liver sections were highly correlated with measured values and more accurate than qualitative copper scores, which should improve diagnostic usefulness of hepatic copper concentrations and assessments in sequential biopsy specimens.
Objective—To determine total glutathione (GSH) and
glutathione disulfide (GSSG) concentrations in liver
tissues from dogs and cats with spontaneous liver
Sample Population—Liver biopsy specimens from
63 dogs and 20 cats with liver disease and 12 healthy
dogs and 15 healthy cats.
Procedure—GSH was measured by use of an enzymatic
method; GSSG was measured after 2-vinylpyridine
extraction of reduced GSH. Concentrations were
expressed by use of wet liver weight and concentration
of tissue protein and DNA.
Results—Disorders included necroinflammatory liver
diseases (24 dogs, 10 cats), extrahepatic bile duct
obstruction (8 dogs, 3 cats), vacuolar hepatopathy (16
dogs), hepatic lipidosis (4 cats), portosystemic vascular
anomalies (15 dogs), and hepatic lymphosarcoma
(3 cats). Significantly higher liver GSH and protein
concentrations and a lower tissue DNA concentration
and ratio of reduced GSH-to-GSSG were found in
healthy cats, compared with healthy dogs. Of 63 dogs
and 20 cats with liver disease, 22 and 14 had low liver
concentrations of GSH (µmol) per gram of tissue; 10
and 10 had low liver concentrations of GSH (nmol) per
milligram of tissue protein; and 26 and 18 had low
liver concentrations of GSH (nmol) per microgram of
tissue DNA, respectively. Low liver tissue concentrations
of GSH were found in cats with necroinflammatory
liver disease and hepatic lipidosis. Low liver concentrations
of GSH per microgram of tissue DNA
were found in dogs with necroinflammatory liver disease
and cats with necroinflammatory liver disease,
extrahepatic bile duct occlusion, and hepatic lipidosis.
Conclusions and Clinical Relevance—Low GSH values
are common in necroinflammatory liver disorders,
extrahepatic bile duct occlusion, and feline
hepatic lipidosis. Cats may have higher risk than dogs
for low liver GSH concentrations. (Am J Vet Res
Objective—To determine whether metal concentrations in canine liver specimens were influenced by specimen size, assay variability, tissue processing (formalin fixation and deparaffinization), or storage in paraffin blocks.
Sample Population—Liver specimens (fresh frozen and deparaffinized) from 2 dogs with chronic hepatitis (high copper but unremarkable iron concentration [liver 1] and unremarkable copper but high iron concentration [liver 2]) as well as fresh and deparaffinized-archived liver specimens from 20 dogs with various hepatopathies.
Procedures—Fresh frozen liver specimens (obtained via simulated needle-core and wedge biopsy), fresh hepatic tissue, and deparaffinized-archived specimens (0.5 to 14 years old) were analyzed for concentrations of copper, iron, and zinc by atomic absorption flame spectrometry. Clinical severity scores were assigned on the basis of tissue metal concentrations.
Results—Interassay variation of metal standards was < 4%. Measurements of liver tissues on 8 consecutive days yielded high coefficients of variation (3.6% to 50%) reflecting heterogenous histologic metal distribution; variation was highest in liver 1 and deparaffinized-archived tissues. Heterogenous metal distribution was confirmed by histologic evaluation. The largest range of metal concentrations was detected in wedge biopsy specimens. In tissues with high metal concentrations, copper and iron concentrations were significantly lower in needle-core versus wedge biopsy specimens. A higher zinc concentration in deparaffinized-archived specimens masked a low zinc concentration in fresh liver tissue of 10 of 20 (50%) dogs.
Conclusions and Clinical Relevance—Retrospective measurement of copper and iron concentrations but not zinc concentrations in deparaffinized-archived liver specimens provided relevant information. The value of needle-core biopsy specimens for measurement of metal concentrations is questionable.
Objective—To investigate the influence of dietary supplementation with l-carnitine on metabolic rate, fatty acid oxidation, weight loss, and lean body mass (LBM) in overweight cats undergoing rapid weight reduction.
Procedures—Cats fattened through unrestricted ingestion of an energy-dense diet for 6 months were randomly assigned to 4 groups and fed a weight reduction diet supplemented with 0 (control), 50, 100, or 150 μg of carnitine/g of diet (unrestricted for 1 month, then restricted). Measurements included resting energy expenditure, respiratory quotient, daily energy expenditure, LBM, and fatty acid oxidation. Following weight loss, cats were allowed unrestricted feeding of the energy-dense diet to investigate weight gain after test diet cessation.
Results—Median weekly weight loss in all groups was ≥ 1.3%, with no difference among groups in overall or cumulative percentage weight loss. During restricted feeding, the resting energy expenditure-to-LBM ratio was significantly higher in cats that received l-carnitine than in those that received the control diet. Respiratory quotient was significantly lower in each cat that received l-carnitine on day 42, compared with the value before the diet began, and in all cats that received l-carnitine, compared with the control group throughout restricted feeding. A significant increase in palmitate flux rate in cats fed the diet with 150 μg of carnitine/g relative to the flux rate in the control group on day 42 corresponded to significantly increased stoichiometric fat oxidation in the l-carnitine diet group (> 62% vs 14% for the control group). Weight gain (as high as 28%) was evident within 35 days after unrestricted feeding was reintroduced.
Conclusions and Clinical Relevance—Dietary l-carnitine supplementation appeared to have a metabolic effect in overweight cats undergoing rapid weight loss that facilitated fatty acid oxidation.
To characterize clinical features, comorbidities, frequency of bacterial isolation, and survival time in cats with suppurative cholangitis-cholangiohepatitis syndrome (S-CCHS).
168 client-owned cats with S-CCHS.
Data were prospectively (1980 to 2019) collected regarding clinical features, comorbidities, bacterial infection, illness duration, and treatments. Variables were evaluated for associations with survival time.
Median age of cats was 10.0 years, with no breed or sex predilection observed. Common clinical features included hyporexia (82%), hyperbilirubinemia (80%), lethargy (80%), vomiting (80%), jaundice (67%), weight loss (54%), and hypoalbuminemia (50%). Comorbidities included extrahepatic bile duct obstruction (53%), cholelithiasis (42%), cholecystitis (40%), and ductal plate malformation (44%) as well as biopsy-confirmed inflammatory bowel disease (60/68 [88%]) and pancreatitis (41/44 [93%]). Bacterial cultures were commonly positive (69%) despite prebiopsy antimicrobial administration in most cats. Of surgically confirmed choleliths, diagnostic imaging identified only 58%. Among 55 cats with “idiopathic pancreatitis,” 28 (51%) were documented to have transiting choleliths, and 20 had pancreatic biopsies confirming pancreatitis. Cholelithiasis (with or without bile duct obstruction) and cholecystectomy were associated with survival advantages. Survival disadvantages were found for leukocytosis, ≥ 2-fold increased alkaline phosphatase, and hyperbilirubinemia. Cholecystoenterostomy had no survival impact. Cats with ductal plate malformations were significantly younger at diagnosis and death than other cats. Chronic treatments with antimicrobials, S-adenosylmethionine, and ursodeoxycholic acid were common postbiopsy.
S-CCHS in cats was associated with bacterial infection and various comorbidities and may be confused with pancreatitis. Surgically correctable morbidities (ie, cholecystitis, cholecystocholelithiasis) and cholecystectomy provided a significant survival advantage.
To characterize the frequency and type of bacterial infection by culture- and immunohistochemical (IHC)-based methods and determine the impact of infection on clinical features and survival time in cats with suppurative cholangitis-cholangiohepatitis syndrome (S-CCHS).
168 client-owned cats with S-CCHS (cases).
Clinical features, bacterial culture results, culture-inoculate sources, and survival details were recorded. Cases were subcategorized by comorbidity (extrahepatic bile duct obstruction, cholelithiasis, cholecystitis, ductal plate malformation, biopsy-confirmed inflammatory bowel disease, and biopsy-confirmed pancreatitis) or treatment by cholecystectomy or cholecystoenterostomy. Culture results, bacterial isolates, Gram-stain characteristics, and IHC staining were compared among comorbidities. Lipoteichoic acid IHC staining detected gram-positive bacterial cell wall components, and toll-like receptor expression IHC reflected pathologic endotoxin (gram-negative bacteria) exposure.
Clinical features were similar among cases except for more frequent abdominal pain and lethargy in cats with positive culture results and pyrexia, abdominal pain, and hepatomegaly for cats with polymicrobial infections. Bacteria were cultured in 93 of 135 (69%) cats, with common isolates including Enterococcus spp and Escherichia coli. IHC staining was positive in 142 of 151 (94%) cats (lipoteichoic acid, 107/142 [75%]; toll-like receptor 4, 99/142 [70%]). With in-parallel interpretation of culture and IHC-based bacterial detection, 154 of 166 (93%) cats had bacterial infections (gram-positive, 118/154 [77%]; gram-negative, 111/154 [72%]; polymicrobial, 79/154 [51%]). Greater frequency of bacterial isolation occurred with combined tissue, bile, and crushed cholelith inoculates. Infection and gram-positive bacterial isolates were associated with significantly shorter long-term survival times.
S-CCHS was associated with bacterial infection, pathologic endotoxin exposure, and frequent polymicrobial infection in cats. Combined tissue inoculates improved culture detection of associated bacteria.
Objective—To evaluate the influence of a 1,4-
butanedisulfonate stable salt of S-adenosylmethionine
(SAMe) administered orally on clinicopathologic
and hepatic effects induced by long-term administration
of prednisolone in dogs.
Animals—12 healthy dogs.
Procedure—Following a pilot study (4 dogs), 2 groups
of 4 dogs received prednisolone (2.2 mg/kg) orally
once daily (84-day trial). One group received SAMe
(20 mg/kg/d divided in 2 doses) for 42 days and then
a placebo for 42 days; the other group received treatments
in the reverse order. Before and during the
trial, numerous variables were monitored, including
serum total alkaline phosphatase (ALP) and glucocorticoid-
induced ALP (G-ALP) activities, serum haptoglobin
concentration, and total and oxidized glutathione
(TGSH and GSSG) and thiobarbiturate-reacting
substances (TBARS) concentrations in erythrocytes
and liver tissue (days 0, 42, and 84). Hepatic
specimens also were examined microscopically.
Results—The stable salt of SAMe was biologically
available; plasma concentrations of SAMe or prednisolone
were not affected by coadministration.
Compared with baseline values, serum ALP and GALP
activities and haptoglobin concentrations
increased and erythrocyte GSSG and TBARS concentrations
decreased with both treatments. Erythrocyte
TGSH concentration decreased with the prednisolone-
placebo treatment. Administration of SAMe
appeared to conserve erythrocyte TGSH values and
did not inhibit hepatocyte glycogen vacuolation but
increased hepatic TGSH concentration and improved
the hepatic tissue GSSG:TGSH ratio.
Conclusions and Clinical Relevance—In dogs,
administration of 20 mg of SAMe/kg/d may mitigate
the apparent pro-oxidant influences of prednisolone
but did not block development of classic clinicopathologic
or histologic features of vacuolar hepatopathy.
(Am J Vet Res 2005;66:330–341)
Objective—To assess the influence of meal ingestion and orally administered erythromycin on gallbladder volume in dogs.
Animals—22 healthy dogs.
Procedures—Ultrasonographically determined gallbladder dimensions in unsedated dogs were used to calculate volume. Measurements were recorded after food was withheld for 12 hours (time 0) and 15, 30, 45, 60, 90, and 120 minutes after a 100-g meal without (n = 22) or with erythromycin (1.0 mg/kg , 2.5 mg/kg , and both dosages ). Gallbladder ejection fraction represented the percentage of volume change from time 0. Intraday and interday coefficients of variation determined operator repeatability and physiologic variation.
Results—We did not detect significant differences in gallbladder volume per unit of body weight between treatments at time 0 or in ejection fraction percentage within or between treatments. Median time 0 gallbladder volume was 0.6 mL/kg (range, 0.4 to 1.9) but was > 1.0 mL/kg in 3 of 22 (14%) dogs and ≤ 1.0 mL/kg in 19 of 22 (86%) dogs. Twenty dogs achieved an ejection fraction ≥ 25% with at least 1 treatment, but 2 dogs with a gallbladder volume ≤ 1.0 mL/kg at time 0 did not. Intraday and interday coefficients of variation were 18% and 25%, respectively.
Conclusions and Clinical Relevance—Gallbladder volume ≤ 1.0 mL/kg at time 0 and ejection fraction ≥ 25% were typical. No treatment consistently induced greater gallbladder contraction. Dogs with a gallbladder volume > 1.0 mL/kg and ejection fraction < 25% may require a combined meal and erythromycin protocol.
OBJECTIVE To establish reference limits for hepatic bile duct-to-arteriole ratio (BD:A) and bile duct-to-portal tract ratio (BD:PT) in healthy cats and assess whether these parameters could be used to support a diagnosis of biliary ductopenia in cats.
SAMPLE Hepatic biopsy samples from healthy cats (n = 20) and cats with ductopenia (2).
PROCEDURES Hepatic biopsy samples from healthy cats were used to count the number of bile ducts and hepatic arterioles in 20 portal tracts for each cat. Mean BD:A and mean BD:PT for each cat were calculated, and these values were used to determine reference limits for mean BD:A and mean BD:PT. Results of histologic evaluation, including immunohistochemical staining in some instances, were compared for healthy cats versus cats with ductopenia.
RESULTS Of the 400 portal tracts from healthy cats, 382 (95.5%) and 396 (99.0%) had BD:A and BD:PT, respectively, ≥ 1.0, with less variability in BD:A. Mean BD:A and BD:PT were markedly lower in both cats with ductopenia, compared with values for healthy cats. However, only mean BD:A for cats with ductopenia was below the reference limit of 0.59.
CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that systematic evaluation of BD:A, with a lower reference limit of 0.59 to define biliary ductopenia in cats, may be a discrete and easily applied morphometric tool to enhance detection of ductopenia in cats. However, application of this ratio required evaluation of ≥ 20 portal tracts with cross-sectioned portal elements to determine a mean BD:A value.