George R. Morrison

Cell heterogeneity within the hepatic lobule: quantitative histochemistry.

This investigation was undertaken to seek quantitative information concerning the relative activity of certain enzymes in hepatic cells in various parts of the lobule. The enzymes selected for study are chiefly those with important nwtabohc roles. It was thought that such infornmtion might bring important new understandlilig of function in the liver lobule and its relationship to the localization of various types of injury. This approach has been made possible by the inicrochemical rnethodls of Lowry (4) which permit analysis of small groups of cells dissected out of frozen dried! sections of tissue. These method!s have been adlaptcd in our laboratory with the collaboration and assistance of Dr. Lowry and have been utilized in the study of liver of normal young male Sprague-Dawley rats weighing from 180 to 230 grams and! maintained! on a diet of Purina chow fed ad libitum. After eight hours of fast, the animals were sacrificed by decapitation. The liver was weighed and a small biopsy from the periphery of the left lateral lobe was droppedl immediately into liquid nitrogen. Sections were cut at 22 in a cryostat at a temperature of -25#{176}C. The tissue sections were then dehydrated! ifl vacuo at -40#{176}C. After dehydration sections were brought to room temperature in vacuo and!, in an air conditioned room, representative areas of the lobule were dissected out at a magnification of 72 x under the dissecting microscope. The areas selected included small Portal triadis surroundled by a rim

Quantitative analysis of regenerating and degenerating areas within the lobule of the carbon tetrachloride-injured liver.

Abstract The livers of rats were studied 48 hours after acute carbon tetrachloride injury in order to determine the degree to which four areas of the lobule participate in both regenerative and degenerative processes and to relate alterations in enzyme activity in each area to these processes. With histological stains, the intralobular distribution of mitotic activity, neutral fat and necrosis were quantitated. Microchemical techniques in conjunction with microdissection of lyophilized sections were used, and determinations were made of total protein, total hemoglobin, total lipid, and of nine enzymes in all four areas of the liver lobule. Regenerative processes were shown to dominate the portal midzone area of the injured lobule while degenerative processes were localized largely in the central area. Within the degenerated central area, activities of alkaline phosphatase, phosphoglucoisomerase, and glucose-6-phosphate dehydrogenase were elevated above control levels, while activities of lactic dehydrogenase, isocitric dehydrogenase, malic dehydrogenase, glutamic dehydrogenase, glutamic-alanine transaminase, and β-hydroxybutyryl-CoA dehydrogenase were decreased. In the portal midzone, a decreased activity of phosphoglucoisomerase and an elevated activity of lactic dehydrogenase were the only alterations in enzyme activities which were interpreted as being related to the regenerative process when correlations were made between the intralobular patterns of enzyme activities and the intralobular patterns of the manifestations of regeneration and degeneration.

Microchemical determination of organic nitrogen with Nessler reagent.

Abstract A method is presented for the determination of as little as 0.02 μg of organic nitrogen using Nessler reagent solution with colorimetry. Details necessary for obtaining reproducible results are presented because several factors must be controlled carefully to avoid a decrease color development and/or turbidity of the final solution.

Cold-acclimatization and intermediary metabolism of carbohydrates.

Abstract The activities of 14 enzymes participating in the intermediary metabolism of carbohydrates have been measured in the livers of rats to study the effects of chronic cold exposure. Based upon significant alterations in the activities of rate-limiting enzymes, it is concluded that an improved capacity for gluconeogenesis is the major change in hepatic carbohydrate metabolism associated with cold-acclimatization. No significant alterations in the activities of enzymes which are rate-limiting for glycolysis, glycogenesis, or glycogenolysis were found in the livers of rats exposed to 4 ° for 28 days.

Hexokinase and glucokinase activities in bile duct epithelial cells and hepatic cells from normal rat and human livers

Abstract In the livers of normal rats and man, bile duct epithelial cells from interlobular portal tracts and hepatic cells from midzonal areas of lobules have been microdissected and separated. These cells were assayed for hexokinase and glucokinase activities. The results indicate that hepatic parenchymal cells have hexokinase and glucokinase activities, while bile duct epithelial cells have little or no glucokinase activity but hexokinase activity greater than that in parenchymal cells.

Fluorometric measurement of phosphoglucoisomerase

Abstract A fluorometric method is described for assaying phosphoglucoisomerase in as little as 0.05 μg of frozen-dried sections of liver. The simplicity of the procedure and the stability of the fluorescence facilitate the measurement of a large number of samples. Several steps in the procedure have been studied in detail.

Fluorometric microdetermination of glycogen.

Abstract A fluorometric method is described which measures as little as 0.02 μg glycogen in tissue using microequipment or 0.2 μg glycogen using standard equipment. The standard curves for these two procedures are linear up to 0.15 and 1.5 μg glycogen, respectively. The coefficient of variation with the proposed fluorometric method averages 0.9% for fresh livers from fed and fasted rats containing 4.1 to 0.8 gm glycogen per 100 gm tissue. When compared to a popular colorimetric anthrone method, the standard error of mean differences for the proposed fluorometric method was less than 1% of the quantity of glycogen in the average sample. The proposed fluorometric procedure is more specific for glycogen than the anthrone colorimetric method because the fluorescence of G6P, F6P, or FDP is one-half of that produced by an equal quantity of glycogen.