Small Liver Biopsy Research Articles

Role of arginase 1 immunohistochemical marker in differentiating hepatocellular carcinoma from other primary and secondary carcinomas of the liver, a tissue microarray study

: Hepatocellular carcinoma (HCC) is the most prevalent type of primary cancer of the liver in adults represent about 80%-90% of all liver cancers. It is essential to differentiate primary HCC and intrahepatic cholangiocarcinoma and metastatic carcinoma. Arginase-1 was considered as the most sensitive and specific marker of benign and malignant hepatocyte. This study aimed to detect the diagnostic role of immunohistochemical expression of arginase-1 in differentiating HCC From cholangiocarcinoma and metastatic carcinomas of the liver in comparison with hepatocyte paraffin antigen -1 (HepPar-1) and Glypican 3. : This is a retrospective study was performed on 117 cases, 77 cases were diagnosed as HCC, 13 cases as cholangiocarcinoma and 27 cases as metastatic carcinomas in the liver. Cases obtained from surgical pathology laboratory at Gastroenterology Center, Mansoura University, Egypt during the period from 2014 to 2017. All the studied cases were immunostained with Arginase 1, Heppar 1and Glypican 3.: Arginase 1 was expressed in all 77 HCC cases with sensitivity (100%), while Arginase 1 was expressed only in 1 cholangiocarcinoma case and negative in other metastatic carcinomas with specificity(97.5%), the overall accuracy was (99.1%). On the other side, Glypican 3 was expressed in 36 out of 77 HCC cases with sensitivity (46.8%), while Glypican 3 was expressed in 3 out of 40 cholangiocarcinoma and other metastatic carcinomas with specificity (92.3%), overall accuracy (62.4%). Heppar 1 was expressed in 69 out of 77 HCC cases with sensitivity (89.6%), while Heppar 1 was negative in all cholangiocarcinoma cases and other metastatic carcinomas with specificity (100%). The overall accuracy was 93.2%.: Arginase 1 is the most sensitive and accurate marker in differentiating HCC from non HCC cases in liver, while heppar is most specific and second accurate marker in differentiating HCC from cholangiocarcinoma and metastatic carcinomas in the liver. Arginase-1 and HepPar-1, are the best markers regarding sensitivity and specificity for small liver biopsies.

Correlation between Islet Number in the Small Biopsy Liver and Blood Glucose Level after Pig to Non-Human Primate Islet Xenotransplantation Model

Introduction Pig to non-human primate (NHP) islet xenotransplantation is currently being explored as a pre-clinical model for treatment of type 1 diabetes patients with hypoglycemia unawareness. Pig islet has been shown to regulate blood glucose control in diabetic NHPs. However, due to initial immune responses including IBMIR etc., a large number of islets are destroyed and subsequently the remaining islets can engraft and regulate blood glucose level in the recipients. Among these pre-clinical studies, the proper number of islets required for blood glucose level control in diabetic NHPs has not been reported. Here, we measure the number of islets present in small biopsy liver samples from NHPs underwent porcine islet transplantation and correlate its number with blood glucose levels during follow-up periods. Method Pig islets were isolated from designated pathogen-free SNU miniature pigs and transplanted via a portal vein into streptozotocin-induced diabetic monkeys (Rhesus macaque). After transplantation, 42 biopsies were performed from 29 NHPs. The experimental group was divided into three groups; normoglycemia (about 100-150 mg/dl), intermediate hyperglycemia (150-250 mg/dl) and hyperglycemia (more than 250 mg/dl) according to blood glucose levels. To measure the number of transplanted islets, a biopsy of the distal portion of the liver was performed and the biopsied samples were stained with anti-insulin antibodies. Also, semi-quantitative histological and statistical analyses were conducted. Result Pig islets were positively stained with anti-insulin antibody in the liver samples from the recipient NHPs. The number of stained islets per cm2 was measured in liver section, with its number ranging from 0 to 26.6. The values was 3.89-26.6, 1.18-3.77, and 0-2.53 in the normoglycemia, intermediate hyperglycemia, and hyperglycemia group, respectively. Importantly, islet number in the liver section was negatively correlated to the level of fasting blood glucose at the day of biopsy and this relationship is highly significant (Figure 1). Conclusion The number of islets present in small biopsy liver samples is significantly correlated with blood glucose level in islet recipient NHPs. This finding suggests that islet number in the small liver biopsy samples can represent engrafted islet mass and thus be potentially one of parameters to predict the outcome of islet transplantation.This study was supported by a grant from the Korea Healthcare Technology R&D Project, Ministry for Health & Welfare, Republic of Korea (Project No. HI13C0954) and the National Research Foundation of Korea (Project No. NRF-2017R1D1A1B03030897). Anti-CD40 antibody used in these studies was provided by the Nonhuman Primate Reagent Resource supported by the NIH Nonhuman Primate Reagent Resource (U24 AI126683 and R24 OD10976).

Respiratory analysis of coupled mitochondria in cryopreserved liver biopsies

The aim of this work was to develop a cryopreservation method of small liver biopsies for in situ mitochondrial function assessment. Herein we describe a detailed protocol for tissue collection, cryopreservation, high-resolution respirometry using complex I and II substrates, calculation and interpretation of respiratory parameters. Liver biopsies from cow and rat were sequentially frozen in a medium containing dimethylsulfoxide as cryoprotectant and stored for up to 3 months at −80 °C. Oxygen consumption rate studies of fresh and cryopreserved samples revealed that most respiratory parameters remained unchanged. Additionally, outer mitochondrial membrane integrity was assessed adding cytochrome c, proving that our cryopreservation method does not harm mitochondrial structure. In sum, we present a reliable way to cryopreserve small liver biopsies without affecting mitochondrial function. Our protocol will enable the transport and storage of samples, extending and facilitating mitochondrial function analysis of liver biopsies.

Technical note: Analysis of total lipid and triacylglycerol content in small liver biopsy samples in cattle1

A procedure is described for analyzing total lipid (TL) and triacylglycerol (TAG) in 2 sequential steps using small amounts (<100 mg) of bovine liver tissue. The TL was measured gravimetrically and TAG was measured enzymatically in the TL extract, using an automated analyzer. For gravimetric TL determination in milligrams per gram of liver fresh weight (FW), TL was extracted from homogenized tissue samples with hexane:isopropanol (at 20 degrees C, 24 h, constant agitation). The routine method was modified by adding a second hexane extraction step to optimize lipid extraction. The dry lipid extract was dissolved in hexane and aliquoted according to TL content for TAG analysis. An extra incubation period of 16 h was included for complete hydrolysis of TAG, using microbial lipase and nonaethylene glycol monododecyl ether detergent, before TAG was measured enzymatically using commercial test kits. Triolein was used as an internal standard. Repeated TL analysis (n = 3) of liver specimens from 10 cows (range, 40 to 314 mg/g of FW) yielded a mean CV of 2.2%, whereas repeated TAG analysis (range, 4 to 260 mg/g of FW) yielded a mean intraday CV of 2.5% (n = 5) and a mean interday CV of 3.4% (n = 4). Intraday (n = 5) and interday (n = 4) CV for repeated TAG analysis in triolein standards were <1 and <3%, respectively. Recovery of TAG in triolein standards varied between 99 and 103%. In part 2 of the experiment, hepatic TL and TAG were measured in 150 German Holstein cows to verify the test method in a large sample size. For repeated hepatic TL (n = 3) and TAG (n = 5) determination, mean CV of <2.8 and <1.5%, respectively, were found. The proportion of TAG relative to TL increased linearly to a breakpoint of approximately 100 mg TL/g of FW, at which point it reached a plateau at approximately 68%, indicating an accumulation of other lipid fractions in hepatic tissue with hepatic TL above the breakpoint. Calculation of hepatic TAG from TL was reasonably accurate when a 2-slope linear broken-line model (r(2) = 0.98) was used. Above a TL of approximately 40 mg/g of FW, calculated TAG values deviated by only +/-15% from measured hepatic TAG.

Comparison of different methods to obtain and store liver biopsies for molecular and histological research

BackgroundTo minimize the necessary number of biopsies for molecular and histological research we evaluated different sampling techniques, fixation methods, and storage procedures for canine liver tissue. For addressing the aim, three biopsy techniques (wedge biopsy, Menghini, True-cut), four storage methods for retrieval of RNA (snap freezing, RNAlater, Boonfix, RLT-buffer), two RNA isolation procedures (Trizol and RNAeasy), and three different fixation protocols for histological studies (10% buffered formalin, RNAlater, Boonfix) were compared. Histological evaluation was based on hematoxylin-eosin (HE) and reticulin (fibrogenesis) staining, and rubeanic acid and rhodanine stains for copper. Immunohistochemical evaluation was performed for cytokeratin-7 (K-7), multidrug resistance binding protein-2 (MRP-2) and Hepar-1.ResultsRNA quality was best guaranteed by the combination of a Menghini biopsy with NaCl, followed by RNAlater preservation and RNAeasy mini kit extraction. These results were confirmed by quantitative RT-PCR testing. Reliable histological assessment for copper proved only possible in formalin fixed liver tissue. Short formalin fixation (1–4 hrs) improved immunohistochemical reactivity and preservation of good morphology in small liver biopsies.ConclusionAt least two biopsies (RNAlater and formalin) are needed. Since human and canine liver diseases are highly comparable, it is conceivable that the protocols described here can be easily translated into the human biomedical field.

CD56 expression aids in the differential diagnosis of cholangiocarcinomas and benign cholangiocellular lesions

CD56 (neuronal cell adhesion molecule, N-CAM) has been reported in neuroendocrine tumours and as a marker of reactive biliary epithelial cells. However, up to date, it is not used to distinguish malignant from non-malignant biliary lesions. In this study, we systematically examined CD56 expression on 98 tumours arising from the biliary tree as well as intrahepatic conditions with reactive neoductules. When neuroendocrine carcinomas are excluded, only 4 of 32 (12.5%) cholangiocarcinomas expressed CD56, 2 of which showed clear cell morphology. Reactive bile ductules adjacent to cirrhotic nodules as well as in focal nodular hyperplasia were CD56 positive. Twelve of 17 (70.5%) bile duct adenomas were CD56 positive, whereas von Meyenburg complexes expressed CD56 only very focally in less than 5% of lesional cells. Bile duct cysts were negative for CD56 with the exception of focally interspersed neuroendocrine cells, similar to that seen in segmental bile ducts. Thus, if van Meyenburg complexes are excluded, CD56 can be used to differentiate intrahepatic non-neoplastic from neoplastic proliferations, which is a helpful diagnostic tool in small liver biopsies.

Proteome Analysis of Liver Cells Expressing a Full-Length Hepatitis C Virus (HCV) Replicon and Biopsy Specimens of Posttransplantation Liver from HCV-Infected Patients

The development of a reproducible model system for the study of hepatitis C virus (HCV) infection has the potential to significantly enhance the study of virus-host interactions and provide future direction for modeling the pathogenesis of HCV. While there are studies describing global gene expression changes associated with HCV infection, changes in the proteome have not been characterized. We report the first large-scale proteome analysis of the highly permissive Huh-7.5 cell line containing a full-length HCV replicon. We detected >4,200 proteins in this cell line, including HCV replicon proteins, using multidimensional liquid chromatographic (LC) separations coupled to mass spectrometry. Consistent with the literature, a comparison of HCV replicon-positive and -negative Huh-7.5 cells identified expression changes of proteins involved in lipid metabolism. We extended these analyses to liver biopsy material from HCV-infected patients where a total of >1,500 proteins were detected from only 2 mug of liver biopsy protein digest using the Huh-7.5 protein database and the accurate mass and time tag strategy. These findings demonstrate the utility of multidimensional proteome analysis of the HCV replicon model system for assisting in the determination of proteins/pathways affected by HCV infection. Our ability to extend these analyses to the highly complex proteome of small liver biopsies with limiting protein yields offers the unique opportunity to begin evaluating the clinical significance of protein expression changes associated with HCV infection.

Pathology of portal hypertension

Abstract Portal hypertension may be the result of intra‐ and extrahepatic impediment of portal venous blood flow. Portal vein thrombosis is the major cause of prehepatic portal hypertension, and hepatic vein occlusion, congestive heart failure and constrictive pericarditis lead to posthepatic portal hypertension. Cirrhosis is the most common intrahepatic lesion responsible for portal hypertension. Idiopathic non‐cirrhotic portal hypertension results from obliterative injuries more specifically directed to intrahepatic portal vascular structures (macro‐ and microvascular portal vein disease). Under these conditions, portal vascular dilatation, herniation into the periportal parenchyma and paraportal shunt vessels resemble collateral pathways. The liver parenchyma often shows nodular transformation which may even be the only clue to the presence of small portal vein obstruction in a small liver biopsy. Thus, the morphologic changes associated with idiopathic portal hypertension may be subtle and non‐specific. Their recognition, particularly in the liver biopsy, is a challenge to the pathologist.

Overexpression of interferon γ-inducible protein 10 in the liver of patients with type i autoimmune hepatitis identified by suppression subtractive hybridization

To clarify gene expression profiles in the liver may elucidate the pathogenesis of type I autoimmune hepatitis (AIH). Using suppression subtractive hybridization (SSH), we identified genes overexpressed in the liver of AIH. A small liver biopsy sample from a patient with definite AIH was available to be analyzed in our system. By mixing cDNA synthesized from this sample as a 'tester' and cDNA from a normal liver as a 'driver,' we subtracted cDNA to enrich genes overexpressed in AIH. After polymerase chain reaction (PCR) amplification and subcloning, we identified subtracted genes by sequencing 50 randomly selected clones. Only one cDNA fragment, which is identical to interferon inducible protein 10 (IP-10), was overexpressed by > 10 times in the liver of AIH, as compared with control. We confirmed IP-10 overexpression in all eight patients with AIH by reverse transcription PCR. Immunohistochemical analysis demonstrated increased IP-10 expression in hepatocytes in the liver of AIH. Reverse transcription PCR analysis of 63 liver biopsy samples with various liver diseases revealed that IP-10 expression was significantly higher in AIH (p = 0.025) and chronic hepatitis C (p = 0.0043) than in other liver diseases. Interestingly, the amount of IP-10 mRNA expression was correlated with serum ALT values in AIH (p = 0.0006), but not in chronic hepatitis C (p = 0.43). These results indicate the IP-10 expression in the liver might be used as a preferential marker of AIH, and that IP-10 has some pathophysiological roles in the liver damage of AIH.

Overexpression of interferon γ-inducible protein 10 in the liver of patients with type i autoimmune hepatitis identified by suppression subtractive hybridization

OBJECTIVE: To clarify gene expression profiles in the liver may elucidate the pathogenesis of type I autoimmune hepatitis (AIH). Using suppression subtractive hybridization (SSH), we identified genes overexpressed in the liver of AIH. METHODS: A small liver biopsy sample from a patient with definite AIH was available to be analyzed in our system. By mixing cDNA synthesized from this sample as a ‘tester’ and cDNA from a normal liver as a ‘driver,’ we subtracted cDNA to enrich genes overexpressed in AIH. After polymerase chain reaction (PCR) amplification and subcloning, we identified subtracted genes by sequencing 50 randomly selected clones. RESULTS: Only one cDNA fragment, which is identical to interferon inducible protein 10 (IP-10), was overexpressed by >10 times in the liver of AIH, as compared with control. We confirmed IP-10 overexpression in all eight patients with AIH by reverse transcription PCR. Immunohistochemical analysis demonstrated increased IP-10 expression in hepatocytes in the liver of AIH. Reverse transcription PCR analysis of 63 liver biopsy samples with various liver diseases revealed that IP-10 expression was significantly higher in AIH ( p = 0.025) and chronic hepatitis C ( p = 0.0043) than in other liver diseases. Interestingly, the amount of IP-10 mRNA expression was correlated with serum ALT values in AIH ( p = 0.0006), but not in chronic hepatitis C ( p = 0.43). CONCLUSION: These results indicate the IP-10 expression in the liver might be used as a preferential marker of AIH, and that IP-10 has some pathophysiological roles in the liver damage of AIH.

Metabolic capacities in cultured human hepatocytes obtained by a new isolating procedure from non-wedge small liver biopsies

Metabolic capacities in cultured human hepatocytes obtained by a new isolating procedure from non-wedge small liver biopsies

Characterisation of the DNA repair enzyme for O6-methylguanine in cirrhosis

Background/Aims: The reason(s) why cirrhosis is a major risk for the development of hepatocellular carcinoma worldwide are largely unknown. In a previous preliminary study, we reported deficiency of the repair enzyme for the highly promutagenic and potentially carcinogenic DNA base lesion, O 6-methylguanine, in cirrhotic human liver. The aims of the present study were: (i) to confirm this observation in an extended series of cirrhosis patients, using a new DNA repair enzyme assay approach, (ii) to characterise the enzyme, in particular to seek physicochemical differences between control and cirrhotic liver that might account for the enzyme defect incirrhosis, and (iii) to examine the relationship between magnitude of DNA Repair deficiency incirrhotic liver and aetiology of cirrhosis or male sex, both of which are independent risk factors for hepatocellular carcinoma. Methods: Tissue extracts of containing DNA repair enzyme, O 6-methylguanine-DNA methyltransferase, were prepared from liver biopsy samples from 41 patients with cirrhosis (10 viral (HBV and HCV), 17 alcohol and 14 autoimmune), 17 patients with non-cirrhotic diseased liver and 4 patients with histologically normal liver. Repair enzyme levels and electrophoretic profiles were both obtained using sodium dodecyl sulphate-polyacrylamide gel electrophoresis in conjunction with fluorography and densitometry. Results: We have demonstrated the feasibility of both analysing and reproducibly measuring DNA repair enzyme in small liver biopsy samples using the gel electrophoresis/densitometry approach for enzyme assay. Using the new densitometric assay approach, levels of O 6-methylguanine-DNA methyltransferase were found to be significantly lower in cirrhotic compared to non-cirrhotic liver, with very low values obtained for two individuals who were incidentally found to have small hepatocellular carcinomas at the time of liver transplantation. There were no significant differences in enzyme levels between patients with cirrhosis of different aetiology, and between male and female patients. Methyltransferase was found to be present in all liver extracts as a major 23.1 kDa protein, along with other less abundant enzyme forms of both slightly higher and lower molecular weight; there were no obvious differences in size and relative abundance of these enzyme forms between liver samples from any of the patient and control groups. Our findings of enzyme instability and multiple forms led us to examine methyltransferase's amino acid sequence for the presence of primary structure motifs indicative of targeted degradation of the enzyme by intracellular proteases. We have identified a eukaryotic thiol protease active sige motif and two cyclin-like “destruction bos” motifs in the N-terminal half of the enzyme. Conclusions: Our findings provide further evidence suggesting that deficiency in the ability to repair O 6-methylguanine-DNA underlies the increased risk of hepatocellular carcinoma seen in patients with cirrhosis. The degree of DNA repair deficiency not varying between cirrhotic patients subgrouped according to other risk factors for hepatocellular carcinoma, suggests that DNA repair deficiency is only one of several contributory factors in liver carcinogenesis. The presence in methyltransferase of thiol protease active site and “destruction box” motifs may be of relevance to observed repair enzyme instability and presence of multiple enzyme forms in human liver extracts.

Functional human hepatocytes: isolation from small liver biopsy samples and primary cultivation with liver-specific functions.

Morphologically and functionally intact human hepatocytes were isolated from small liver biopsy samples weighing about 1-2 g by initial digestion with collagenase followed by repeated digestions with trypsin. The usual yield of hepatocytes was greater than 1 x 10(7) cells per g of liver sample and cell viability, as judged by dye exclusion test, was routinely over 90%. The isolated human hepatocytes showed intact morphology under scanning electron microscope. Formation of membrane protrusions upon phalloidin addition demonstrated that the actin in isolated hepatocytes was maintained with its structural integrity. The cultured human hepatocytes retained a variety of liver-specific functions which were similarly exhibited by rat hepatocytes isolated using the same procedure. The cultured human hepatocytes exhibited a specific cytochrome P-450 related enzyme activity, and active amino acid uptake that increased upon addition of hormones like glucagon and dexamethasone. Additionally, the cultured human hepatocytes synthesized DNA actively and, human serum albumin, and was found to be responsive to modulation by growth modulating hormones, cytokines and hepatotoxic agents. Based on the profile of activity described above, the presently established conditions for isolation and culturing of human hepatocytes demonstrate that functional liver cells can be obtained from small biopsied liver samples.

RT-PCR detection of fibronectin EDA+ and EDB+ mRNA isoforms: molecular markers for hepatocellular carcinoma.

Alternative splicing of fibronectin pre-mRNA has been shown to be independently regulated at the EDA and EDB regions in a tissue and developmental stage-specific manner. In this study, RT-PCR approaches were developed for the detection of EDA and EDB FN mRNA isoforms in hepatocarcinoma cells (SK-Hep-I) grown in vitro and in human liver biopsies. While EDA+ and EDB+ isoforms were not present in control adult liver, they were detectable in the hepatocarcinoma cells and in fetal liver. The RT-PCR analysis, extended to biopsies of malignant and non-malignant hepatic tissues, showed that FN mRNAs containing the EDA and EDB sequences were present in the 14 hepatocellular carcinomas (HCCs) tested but absent in the non-tumorous liver tissues (i.e., normal parenchyma, non-specific reactive and chronic hepatitis, steatosis). The EDB+ FN mRNA isoforms were also detected in 3 cases of benign neoplasm (hepatocellular adenoma, HCA, I; nodular focal hyperplasia, NFH, 2), while the EDA+ was only detectable in I of the 2 cases of NFH. In addition, both EDA+ and EDB+ isoforms were expressed in 5 out of 9 cirrhotic livers surrounding the tumors. This molecular analysis, which can also be performed on small liver biopsies (2 mg), may therefore be a useful additional tool in the diagnosis of HCC.

Studies of the Cause and Treatment of Hyperammonemia in Females With Ornithine Transcarbamylase Deficiency

Assay of ornithine transcarbamylase (OTC) activity in multiple small bits of liver (approximately 5 mg) that were obtained from a single surgical biopsy in a patient with OTC deficiency revealed a 10- to 40-fold variation in enzyme activity. Similar studies with control autopsy liver specimens varied 2.5-fold at most. The greater variation in the patient with OTC deficiency probably is due to sampling of clusters of normal or abnormal hepatocytes that resulted from inactivation of either the abnormal or normal X chromosone. Enzyme activity assayed on small liver biopsy specimens may not be representative of the entire liver in female patients with OTC deficiency. The hyperammonemia in individuals heterozygous for OTC deficiency may be due in part to shunting of blood through multiple "metabolic portosystemic shunts." Treatment of a girl who has OTC deficiency with a low-protein diet, a low-protein diet supplemented with oral essential amino acids, and a low-protein diet plus oral ketoacids of essential amino acids, on a separate occasion, a low-protein diet was compared to a low-protein diet plus lactulose. The low-protein diet plus oral ketoacid supplementation resulted in the best metabolic control of the patient's disease. On the other hand, paradoxical transient hyperammonemia was observed after the intarvenous administration of ketoacids to two acutely ill female patients with OTC deficiency.

METABOLIC PORTOSYSTEMIC SHUNTS“ IN ORNITHINE TRANS-CARBAMYLASE DEFICIENCY?

Females with ornithine transcarbamylase (OTC) deficiency, an X-linked disorder, are mosaic in that they have hepatocytes with almost no enzyme activity and hepatocytes with normal enzyme activity. The normal and abnormal cells occur in clusters since cells in an area tend to be the progeny of the same parent cell. Marked variation in OTC was found in small liver biopsies from a female with OTC deficiency, probably because the samples included clusters of normal or abnormal cells. OTC activity was measured in 10 approx. 5 mg specimens of liver from a single surgical biopsy from a 7 year old girl with OTC def. (two experiments and control autopsy liver (three experiments). The OTC activity in the patient varied 10-40 fold in the two experiments with a coefficient of variation of 76 and 97 percent (control 20,21 and 26 percent). Thus OTC activity in a small biopsy in females with OTC def. may not be representative of the entire liver. The OTC activity in several bits of liver from our patient was very low suggesting that clusters of abnormal cells may often involve a major portion of a hepatic lobule. If so, blood passing through these areas would in effect be passing through a “metabolic shunt.” These shunts may, in part, account for the hyperammonemia in this disorder. The notion that an X-linked disorder can produce disease in heterozygotes by affecting small functional units could apply to other disorders such as nephrogenic diabetes insipidus or vitamin D dependent rickets.

Glucose-6-phosphatase assay on microgram amounts of liver tissue

To make possible the assay of glucose-6-phosphatase activity on very small liver biopsy specimens a method was worked out based on the following principles: 1. Utilization of small plastic tubes, enclosed in larger glass tubes, to avoid evaporation of the small (0.02 ml) sample during several hours of incubation. 2. Stabilization of the enzyme by sucrose and EDTA. 3. Determination of liberated phosphate by a newly described, highly sensitive method, using Malachite Green. With this method, 1.56 μg of rat liver tissue in each tube was sufficient for an assay. When more convenient, up to at least 100 μg of tissue could equally well be used. When compared to a reference method, using 1 mg of tissue in each tube, similar values for the glucose-6-phosphatase activity were obtained. The coefficient of variation, when 20 analyses were made on the same homogenate, was 13.3%, as compared to 11.5% for the reference method.