Thromboelastographic results and hypercoagulability in dogs with surgically treated hepatocellular adenoma and carcinoma: A Veterinary Society of Surgical Oncology prospective study.

BACKGROUND: The most common haemostatic abnormality in dogs with cancer is hypercoagulability. A transient hypercoagulability has been documented in people with hepatocellular carcinoma (HCC) that resolves within weeks following hepatic tumour resection. OBJECTIVE: The objective was to compare the haemostatic status of dogs with liver tumours and healthy control dogs, by comparing coagulation and thromboelastography (TEG) measurements at three time points. METHODS: Liver tumour and healthy control dogs receiving surgery for liver lobectomy and ovariohysterectomy, respectively, were prospectively enrolled. All dogs had blood collected at three time points: pre-operative, 24 h post-operative and ~2 weeks post-operative. Haematological and haemostatic values were compared across time points in each group using repeated measures ANOVA tests. RESULTS: Ten and eight dogs were enrolled for the liver and control groups, respectively. Platelet count was significantly higher in the liver group than the control group at all time points, but within the normal range (pre-operative: 438.7 vs. 300.9 × 109 /L, p = .0078; 24 h post-operative: 416.2 vs. 283.9 × 109 /L, p = .0123; 10-14 days post-operative: 524.6 vs. 317.3 × 109 /L, p = .0072). The measure of the overall coagulant state (G-value) was significantly increased for the liver group compared to the control group at all time points (pre-operative: 15.6 vs. 8.6 d/sc, p = .0003; 24 h post-operative: 18.3 vs. 11.2 d/sc, p = .039; 10-14 days post-operative: 15.1 vs. 9.6 d/sc, p = .015). CONCLUSION: The liver group was hypercoagulable based on elevated G-values at all time points compared to the control group. This hypercoagulability was attributed to the effect of hepatic tumours alone, and not secondary to surgery and anaesthesia.

Needle tract seeding and malignant transformation of hepatocellular adenoma into well-differentiated hepatocellular carcinoma in a dog.

An 11-year-old neutered female Golden Retriever was referred for investigation of marked increases in liver enzyme activities. Abdominal ultrasound revealed a large pedunculated liver mass. Diagnosis of hepatocellular adenoma (HCA) was made when the mass was excised after a first unsuccessful attempt through ultrasound-guided core-needle biopsy. One and a half years after presentation, a nodule embedded between muscles of the abdominal wall appeared. The mass was first diagnosed as a well-differentiated hepatocellular carcinoma (HCC) through cytologic examination, which was later confirmed with histopathology. Ki 67 immunostaining of the abdominal wall nodule showed an increased immunoreactivity compared with the liver mass. Therefore, the present case documents the first needle-tract seeding of a hepatocellular epithelial tumor with possible malignant transformation of HCA into a well-differentiated HCC in a dog.

RETROSPECTIVE EVALUATION OF HEPATOCELLULAR NEOPLASMS IN NILE LECHWE (KOBUS MEGACEROS) FROM TWO FLORIDA ZOOLOGICAL INSTITUTIONS.

This case series describes hepatocellular neoplasms in 10 Nile lechwe (Kobus megaceros) at two separate zoological institutions in Florida. Histologically, the neoplasms were classified as hepatocellular carcinoma (n = 7), hepatocellular adenoma (n = 2), and hepatobiliary carcinoma (n = 1). Common clinical signs were nonspecific and included thin body condition (n =7), lethargy (n =6), lameness (n =3), and acute recumbency (n =5). Four males and six females were affected, and the mean age at death was 12.7 yr with a range of 4-18 yr. All cases were diagnosed postmortem, and metastasis to various sites, including lung, lymph nodes, and omentum, was found in 40% of cases (n = 4). A single case of hepatocellular carcinoma in a Nile lechwe was described in 2007; however, this is the first reported series of neoplasms in Reduncinae. The pathogenesis behind the development of hepatocellular neoplasms in Nile lechwe has not yet been identified.

Evaluation of hepatic tumor portal perfusion using mesenteric angiography: A pilot study in 5 dogs.

BACKGROUND: Mesenteric angiography is a sensitive method for visualizing portal perfusion in the dog. OBJECTIVES: To evaluate hepatic portal perfusion in dogs with incompletely resectable hepatic tumors using mesenteric angiography. ANIMALS: Five client-owned dogs with incompletely resectable hepatic tumors evaluated with mesenteric angiography. METHODS: Retrospective case series. Electronic medical records at the Animal Medical Center were analyzed to identify dogs that underwent mesenteric portography to determine blood flow to nonresectable hepatic tumors and subsequently determine ideal routes for transarterial embolization, vascular stent placement, or both. The images obtained from mesenteric angiography were analyzed and compared to those obtained from computed tomography angiography. RESULTS: Portography was accomplished using direct mesenteric venography in 3 dogs with hepatocellular carcinoma (HCC), cranial mesenteric arteriography in 1 dog with hepatic adenoma or well-differentiated HCC, and via splenic arteriovenous fistula in 1 dog with diffuse hepatic hemangiosarcoma metastases. Mean pixel densities in areas of hepatic tumor growth identified statistically significant decreases in portal blood flow (P = .02) compared to normal hepatic parenchyma. CONCLUSIONS AND CLINICAL IMPORTANCE: Initial findings indicate that the blood supply to large and metastatic hepatic tumors in dogs may correlate with that in humans, such that the majority of the tumor blood supply arises from the hepatic artery and not the portal vein. Differences in blood supply between normal hepatic parenchyma and hepatic tumors might be exploited by developing selective tumor therapies such as arterial embolization or chemoembolization that largely spare normal liver tissue. Further investigation is warranted.

Hepatocellular adenoma in a European flatfish (Limanda limanda): Genetic alterations in laser-capture micro-dissected tissue and global transcriptomic approach.

Liver tumours in flatfish have been diagnosed using histopathology for decades to monitor the impacts of marine pollution. Here we describe the application of specific gene (retinoblastoma, Rb) profiling in laser capture micro-dissected samples, and a suppression subtractive hybridization (SSH) approach to isolate differentially expressed genes in hepatocellular adenoma (HCA) samples from dab, Limanda limanda. The Rb profiles from apparently normal and HCA micro-dissected samples of fish from the North Sea showed no significant difference, and genotypic heterogeneity within defined histological phenotypes was observed. In the SSH, sequences associated with cell signalling, cell cycle, gene expression regulation, protein transport and protein degradation were isolated. These included up-regulation of arrestin domain containing 3 (arrdc3), Rac-1 and tribbles, and down-regulation of ankyrin repeat/sterile alpha-motif domain-containing protein 1B-like (ANKS1B-like), c-fos, CDKN1B and RhoA-like sequences, previously implicated in mammalian HCA. This study offers new candidates involved in fish liver tumour development.

Neoplasmas em suínos: 37 casos / Neoplasia in swine: 37 cases

Neoplasmas em suínos são raros. Esse trabalho descreve os neoplasmas encontrados em suínos na rotina diagnóstica de um laboratório de patologia veterinária localizado na Região Central do Rio Grande do Sul. Durante um período de 49 anos, 2.266 casos de várias afecções em suínos foram encontrados, dos quais 37 (1,6%) eram neoplasmas. Em ordem decrescente de frequência, os seguintes neoplasmas foram encontrados: Linfoma (11/37), nefroblastoma (11/37), melanoma (8/37) e papiloma (2/37). Adicionalmente, um caso de cada um dos seguintes tumores foi observado: Adenoma hepatocelular, carcinoma hepatocelular, colangiocarcinoma, histiocitoma fibroso maligno e sarcoma granulocítico. O aspecto macroscópico e histológico desses tumores é descrito e a sua epidemiologia é comparada com os dados disponíveis na literatura para neoplasia suína.

Neoplasms in swine are rare. This paper describes neoplasms found in swine in the diagnostic routine of a veterinary pathology laboratory in the Central Region of Rio Grande do Sul, Brazil during a 49-year period, during which 2,266 cases of the various affections in swine were diagnosed. Of those 37 cases (1.6%) were neoplasms. In decreasing order of prevalence, the following neoplasms were found: lymphoma (11 out of 37 cases), nephroblastoma (11/37), melanoma (8/37), and papilloma (2/37). Adenoma hepatocelular, carcinoma hepatocelular, cholangiocarcinoma, malignant fibrous histiocytoma, granulocytic sarcoma were each found in one case out of the 37 cases. The gross and histological aspects of these tumors are described and their epidemiology is compared with the data available in the literature for neoplasia in swine.

Histopathological assessment of liver and gonad pathology in continental slope fish from the northeast Atlantic Ocean.

The deep-sea environment is a sink for a wide variety of contaminants including heavy metals and organic compounds of anthropogenic origin. Life history traits of many deep-water fish species including longevity and high trophic position may predispose them to contaminant exposure and subsequent induction of pathological changes, including tumour formation. The lack of evidence for this hypothesis prompted this investigation in order to provide data on the presence of pathological changes in the liver and gonads of several deep-water fish species. Fish were obtained from the north east region of the Bay of Biscay (north east Atlantic Ocean) by trawling at depths between 700 and 1400 m. Liver and gonad samples were collected on board ship and fixed for histological processing and subsequent examination by light microscopy. Hepatocellular and nuclear pleomorphism and individual cases of ovotestis and foci of cellular alteration (FCA) were detected in black scabbardfish (Aphanopus carbo). Six cases of FCA were observed in orange roughy (Hoplostethus atlanticus) (n = 50) together with a single case of hepatocellular adenoma. A wide variety of inflammatory and degenerative lesions were found in all species examined. Deep-water fish display a range of pathologies similar to those seen in shelf-sea species used for international monitoring programmes including biological effects of contaminants. This study has confirmed the utility of health screening in deep-water fish for detecting evidence of prior exposure to contaminants and has also gained evidence of pathology potentially associated with exposure to algal toxins.

Disruption of DNA methylation via S-adenosylhomocysteine is a key process in high incidence liver carcinogenesis in fish.

Interactions between epigenome and the environment in biology and in disease are of fundamental importance. The incidence of hepatocellular adenomas in flatfish exceeds 20% in some environments forming a unique opportunity to study environmental tumorigenesis of general relevance to cancer in humans. We report the novel finding of marked DNA methylation and metabolite concentration changes in histopathologically normal tissue distal to tumors in fish liver. A multi-"omics" discovery approach led to targeted and quantitative gene transcription analyses and metabolite analyses of hepatocellular adenomas and histologically normal liver tissue in the same fish. We discovered a remarkable and consistent global DNA hypomethylation, modification of DNA methylation and gene transcription, and disruption of one-carbon metabolism in distal tissue compared to livers of non-tumor-bearing fish. The mechanism of this disruption is linked not to depletion of S-adenosylmethionine, as is often a feature of mammalian tumors, but to a decrease in choline and elevated S-adenosylhomocysteine, a potent inhibitor of DNA methyltransferase. This novel feature of normal-appearing tissue of tumor-bearing fish helps to understand the unprecedentedly high incidence of tumors in fish sampled from the field and adds weight to the controversial epigenetic progenitor model of tumorigenesis. With further studies, the modifications may offer opportunities as biomarkers of exposure to environmental factors influencing disease.

Gene expression analyses of hepatocellular adenoma and hepatocellular carcinoma from the marine flatfish Limanda limanda.

At selected sites around the UK, the offshore sentinel flatfish species dab Limanda limanda are found to contain elevated levels of macroscopic liver tumors. Previous proteomic and metabolomic studies have demonstrated that differences exist between tumor and non-tumor tissues; however, these differing features were not identified, and little is known about the changes at the gene expression level, or whether prognostic markers are present and can be identified. A flounder Platichthys flesus custom cDNA microarray and RT-PCR were used to investigate hepatic mRNA expression in the histologically confirmed tumors, hepatocellular adenoma (HA) and hepatocellular carcinoma (HC) from dab, and in adjacent normal tissue from the same fish. Differences in gene expression were observed between tumor and normal tissues, and between tumor types. A class-prediction approach using 50 transcripts revealed sufficient group-specific expression profiles to allow segregation of samples dependent on their tumor type or the sex of the host. Vitellogenins were found to display the greatest induction (up to 500-fold induction) in some HC tumors from female fish and in both HA and HC tumors from males. To the best of our knowledge, this is the first report of the association of vitellogenin expression with tumors of wild fish.

Hepatocellular adenoma in a Eurasian otter (Lutra lutra).

A 7-year-old female Eurasian otter (Lutra lutra) at the Seoul Grand Park, Korea, died after displaying depression, anorexia, weight loss and rough skin for several days. At necropsy, a solitary friable round mass, which was approximately 12 x 9 x 5 cm and mottled dark red and yellow, was found bulging from the right hepatic lobe. Microscopically, the nonencapsulated, poorly circumscribed mass was composed of solid sheets of neoplastic hepatocytes. In addition, numerous small tan foci, ranging from 0.5 to 1.0 cm in diameter, were evenly scattered throughout the pancreatic tissue. These foci were found to be nonencapsulated, well-demarcated hyperplastic nodules of the exocrine pancreatic gland. We observed neither intrahepatic nor extrahepatic metastases. Based on the gross and microscopic changes, we diagnosed the animal as having a hepatocellular adenoma accompanied by exocrine pancreatic nodular hyperplasia.

Plasma proteome analysis reveals the geographical origin and liver tumor status of Dab (Limanda limanda) from UK marine waters.

The flatfish species dab (Limanda limanda) is the sentinel for offshore marine monitoring in the United Kingdom National Marine Monitoring Programme (NMMP). At certain sites in the North and Irish Seas, the prevalence of macroscopic liver tumors can exceed 10%. The plasma proteome of these fish potentially contains reporter proteins or "biomarkers" that may enable development of diagnostic tests for liver cancer and further our understanding of the disease. Following selection of sample groups by quality-assured histopathology ("phenotype anchoring"), we used surface-enhanced laser desorption/ionization (SELDI) time-of-flight mass spectrometry to produce proteomic profiles of plasma from 213 dab collected during the 2004 UK NMMP. The resulting protein profiles were compared between fish from the North and Irish Seas and between fish with liver neoplasia or nondiseased liver. Significant differences were found between the plasma proteomes of dab from the North Sea and Irish Sea, which in conjunction with artificial neural networks can correctly determine from which sea dab were captured in 85% of the cases. In addition, the presence of liver tumors is associated with significant changes in the plasma proteome. We conclude that SELDI-based plasma profiling is potentially of use in nonlethal marine monitoring using wild sentinels such as dab. Furthermore, accurate selection of sample groups is critical for avoiding effects of confounding factors such as age, gender, and geographic origin of samples.

A biological effects monitoring survey of Cardigan Bay using flatfish histopathology, cellular biomarkers and sediment bioassays: findings of the Prince Madog Prize 2003.

Cardigan Bay on the western coast of the UK is considered a pristine location with much of its coastal and marine habitats protected under various national and EC Directives. Despite this, populations of the flatfish dab (Limanda limanda) captured from Cardigan Bay display elevated levels of liver tumours relative to the background prevalence of the disease. This study describes the findings of a research cruise that took place during November 2003 to assess the prevalence of tumours in dab from selected sites in and around Cardigan Bay. In addition, potential causative mechanisms were investigated via measurement of a range of end points (including composition and abundance of benthic and phytoplankton communities, sediment toxicity and cellular biomarkers of genotoxicity) from sediment, water and biota samples. Fish captured from South Cardigan Bay displayed a relatively higher prevalence of liver tumours compared to those captured from Red Wharf Bay. Hepatocellular adenoma (8% and 2%, respectively) and hepatocellular foci of cell alteration (18% and 6%, respectively) were most prevalent in South Cardigan Bay. Analysis of the sediment failed to distinguish any differences in toxicity between the two sampling sites. However, DNA strand breaks in red blood cells of dab were significantly higher (p < 0.05) in fish collected from Red Warf Bay compared with those sampled at Cardigan Bay. The alignment of biological effects measures via such integrated cruise programs are discussed. This work was partly funded under the auspices of the 2003 Prince Madog Prize.

Liver tumors in wild flatfish: a histopathological, proteomic, and metabolomic study.

Fish play host to viral, bacterial, and parasitic diseases in addition to non-infectious conditions such as cancer. The National Marine Monitoring Programme (NMMP) provides information to the U.K. Government on the health status of marine fish stocks. An aspect of this work relates to the presence of tumors and other pathologies in the liver of the offshore sentinel flatfish species, dab (Limanda limanda). Using internationally agreed quality assurance criteria, tumors and pre-tumors are diagnosed using histopathology. The current study has expanded upon this work by integrating these traditional diagnostic approaches with ones utilizing modern technologies for analysis of proteomic and metabolomic profiles of selected lesions. We have applied SELDI and FT-ICR technologies (for proteomic and metabolomic analyses, respectively) to tumor and non-tumor samples resected from the liver of dab. This combined approach has demonstrated how these technologies are able to identify protein and metabolite profiles that are specific to liver tumors. Using histopathology to classify "analysis groups" is key to the success of such an approach since it allows for elimination of spurious samples (e.g., those containing parasite infections) that may confuse interpretation of "omic" data. As such, the pathology laboratory plays a central role in collating information relating to particular specimens and in establishing sampling groups relative to specific diagnostic questions. In this study, we present pilot data, which illustrates that proteomics and metabolomics can be used to discriminate fish liver tumors and suggest future directions for work of this type.

The gender-specific risk to liver toxicity and cancer of flounder (Platichthys flesus (L.)) at the German Wadden Sea coast.

Flatfish living in coastal areas are chronically exposed to a wide range of toxic and (pro)carcinogenic compounds derived from agriculture and industry. Flounder (Platichthys flesus (L.)) is the main target species for monitoring health effects of contamination in North Sea and Baltic Sea since the species is abundant, benthic, and inhabits shallow waters such as the Wadden Seas and estuaries along salinity gradients into fresh water. Chemical analysis in the same livers as investigated for histopathology in the present study showed positive correlation between accumulation of certain organochlorines in liver and the extend of liver injury. Toxipathic liver changes including neoplasms in female and male flounder were analysed by macroscopic and light microscopic diagnosis during a five-year survey on the basis of internationally accepted criteria agreed upon during the European BEQUALM intercalibration of liver histopathology of flatfish. Hepatocellular carcinogenesis of wild flounder principally showed sequential changes similar to experimental chemical carcinogenesis in other fish species and mammals. These ranged from early foci of altered hepatocytes (vacuolated/clear/eosinophilic, basophilic cells) and the development of adenomas. With progression to hepatocellular carcinomas, livers of wild flounder entered a multistage phase of carcinogenesis comprising of early foci, hepatocellular adenomas and carcinomas, as observed in human liver cancers. Female flounder had three-fold higher frequencies of macroscopically visible neoplasms than males of the same age classes. Histopathological diagnosis showed that hepatocellular alterations in male flounder never developed further than stages of basophilic foci and adenomas, and never into malignancies. In females, tumors of hepatocellular origin clearly dominated, occurred alone and together with cancers of bile duct epithelial cells and endothelial cells (cholangio-carcinomas, angiosarcomas). Because mutations of relevant genes could not be identified in the liver cancers that were analysed in the present study, we conclude that epigenetic events initiate carcinogenesis. Therefore, we conclude that mitogenic carcinogens found in the Elbe estuary play a dominant role for cancer development in this flounder population. Furthermore, the lower availability of reducing power (NADPH) needed for detoxification reactions in females compared to males, together with annual upregulation of the highly potent promotor 17-beta-estradiol during sexual maturation, promote progression of preneoplastic foci of initiated cells to malignant cancers in females.

Hepatocellular adenoma in a 12-year-old crossbred German shepherd dog.

A 12-year-old, neutered male crossbred German shepherd presented with lethargy, inappetence, vomiting, and diarrhea. Bile duct carcinoma was diagnosed by cytological analysis of samples obtained by ultrasound-guided fine-needle aspiration. After surgical excision of the mass, the histologic diagnosis was hepatocellular adenoma.

Immunohistochemical characterization of canine hyperplastic hepatic lesions and hepatocellular and biliary neoplasms with monoclonal antibody hepatocyte paraffin 1 and a monoclonal antibody to cytokeratin 7.

Immunostaining with monoclonal antibody (MoAb) hepatocyte paraffin 1 (Hep Par 1) and an MoAb to cytokeratin 7 (CK7) was performed on 105 formalin-fixed, paraffin-embedded canine hyperplastic and neoplastic hepatic lesions. Hep Par 1 was detected in 12/12 hyperplastic nodules, 17/17 hepatocellular adenomas, and 37/40 hepatocellular carcinomas. The staining was disseminated, granular, and cytoplasmic. This antibody did not react with normal or neoplastic biliary epithelium. Other hepatic tumors or tumors metastatic to the liver did not bind Hep Par 1 except one metastatic intestinal carcinoma. MoAb to CK 7 stained all hyperplastic biliary epithelium and benign cholangiocellular tumors (5/5) and 14/18 cholangiocellular carcinomas. One hepatocellular carcinoma had cells positive for both Hep Par 1 and CK 7. Liver was the only normal tissue tested that reacted with MoAb Hep Par 1. Only five nonhepatic tumors (one adrenocortical carcinoma, one interstitial cell tumor of the testis, one melanoma, and two salivary adenocarcinomas) of 277 tumors tested had focal/multifocal staining for Hep Par 1. Prolonged fixation did not alter the staining with Hep Par 1. We conclude that Hep Par 1 is a specific and sensitive marker for canine hepatocellular tumors and allows distinction between hepatocellular and biliary neoplasms.

cDNA cloning and expression of two Ki-ras genes in the flounder, Platichthys flesus, and analysis of hepatic neoplasms.

The screening of a flounder cDNA library with a partial sequence of ras gene from flounder (exons 1 and 2) allowed the isolation of two complete cDNA sequences (ras1 and ras2) highly homologous to human Ki-rasb genes. ras1 and ras2 sequences have an homology of 77.3% indicating that they represent two distinct genes, which differ particularly in their 3 regions. ras1 and ras2 intron 1 sequencing revealed an homology of only 50%, confirming that they represent two different genes. Both genes encode for a 188 amino-acid protein, a size characteristic of Ki-rasb proteins. ras1 protein has the stronger homology to the human Ki-rasb protein (99% identity) and ras2 presents a 85.5% of homology. Two transcripts of respectively 2 and 2.8 kb were identified by northern blots with either ras1 or 2 probes. Preneoplastic and neoplastic livers collected from 14 flounder did not present any mutation on the ras2 gene.

In situ hybridization demonstration of albumin mRNA in B6C3F1 murine liver and hepatocellular neoplasms.

In situ hybridization was used to detect albumin mRNA in normal liver and hepatocellular neoplasms in 20 male B6C3F1 mice between 17 and 24 months of age. Positive signals for albumin were observed consistently in the cytoplasm of hepatocytes in normal liver, particularly in periportal areas. No signals were observed in other cells, such as Kupffer's cells, mesenchymal cells, or bile duct epithelium. Of hepatocellular adenomas, 11/11 (100%) stained positively for albumin mRNA, whereas 14/15 (93%) of primary hepatocellular carcinomas showed positive expression. Albumin mRNA was also detected in extrahepatic metastases of hepatocellular carcinoma, including 9/15 (60%) of pulmonary neoplasms and 5/12 (42%) of metastases at other sites. The pulmonary metastases of hepatocellular carcinoma frequently exhibited a glandular, papillary, or sarcomatous histologic appearance. The presence of albumin in these tumors, lacking characteristic hepatocellular phenotype, is a potential determinant of hepatic lineage. We conclude that in situ hybridization for albumin mRNA in mice is a useful tool in the differential diagnosis of hepatocellular carcinoma, particularly in the case of pulmonary metastasis. This technique may also enable recognition of hepatocyte differentiation in glandular structures with phenotypic features of biliary cells, as seen in mixed hepatocellular-cholangial neoplasms.

Nonlymphomatous hepatobiliary masses in cats: 41 cases (1972 to 1991).

The medical and necropsy records of 41 cats diagnosed with nonlymphomatous hepatobiliary (NLHB) masses, including neoplasia and cysts, were reviewed. Overall, benign masses (n = 27) were more common than malignant ones (n = 14). The single most common malignancy was cholangiocellular carcinoma. The median age at diagnosis was significantly lower (P < .01) for cats with malignant rather than benign disease. Clinical signs associated with hepatobiliary neoplasia were usually vague and included lethargy, vomiting, and anorexia, often present for at least 2 weeks before presentation. Benign masses were an incidental finding in significantly more (P < .01) of the cases than were malignant masses. Median values for alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin were significantly higher (P < .05) in cats with malignant versus benign masses. The prognosis for malignant disease was poor, with 86% of the cats dying or being euthanatized during hospitalization. Cats with benign disease that underwent exploratory celiotomy were more likely to recover and warranted a more favorable prognosis than cats with malignant tumors. Factors associated with malignancy included age at presentation, presence of clinical signs at presentation, and specific serum chemistry changes.