A new technique employing continuous recirculating perfusion of the rat liver in situ, shaking of the liver in buffer in vitro, and filtration of the tissue through nylon mesh, results in the conversion of about 50% of the liver into intact, isolated parenchymal cells. The perfusion media consist of: (a) calcium-free Hanks' solution containing 0.05% collagenase and 0.10% hyaluronidase, and (b) magnesium and calcium-free Hanks' solution containing 2 mM ethylenediaminetetraacetate. Biochemical and morphologic studies indicate that the isolated cells are viable. They respire in a medium containing calcium ions, synthesize glucose from lactate, are impermeable to inulin, do not stain with trypan blue, and retain their structural integrity. Electron microscopy of biopsies taken during and after perfusion reveals that desmosomes are quickly cleaved. Hemidesmosome-containing areas of the cell membrane invaginate and appear to pinch off and migrate centrally. Tight and gap junctions, however, persist on the intact, isolated cells, retaining small segments of cytoplasm from formerly apposing parenchymal cells. Cells which do not retain tight and gap junctions display swelling of Golgi vacuoles and vacuoles in the peripheral cytoplasm. Cytoplasmic vacuolization in a small percentage of cells and potassium loss are the only indications of cell injury detected. By other parameters measured, the isolated cells are comparable to normal hepatic parenchymal cells in situ in appearance and function.

https://rupress.org/jcb/article-pdf/43/3/506/1069297/506.pdf

HIGH-YIELD PREPARATION OF ISOLATED RAT LIVER PARENCHYMAL CELLS: A Biochemical and Fine Structural Study

Phenol-hypochlorite reaction for determination of ammonia

Automated simultaneous determination of ammonia and total amino acids in ruminal fluid and in vitro media

This Account explores the catalytic reduction of dinitrogen by molybdenum complexes that contain the [HIPTN3N]3- ligand ([HIPTN3N]3- = [(HIPTNCH2CH2)3N]3-, where HIPT = 3,5-(2,4,6-i-Pr3C6H2)2C6H3) at room temperature and pressure with protons and electrons. A total of 7−8 equiv of ammonia is formed out of ∼12 possible (depending upon the Mo derivative employed). No hydrazine is formed. Numerous X-ray studies of proposed intermediates in the catalytic cycle suggest that N2 is being reduced at a sterically protected, single Mo center operating in oxidation states between MoIII and MoVI. Subtle variations of the [HIPTN3N]3- ligand are not as successful as a consequence of an unknown shunt in the catalytic cycle that consumes reduction equivalents to yield (it is proposed) dihydrogen.

https://science.sciencemag.org/content/sci/301/5629/76.full.pdf

Catalytic Reduction of Dinitrogen to Ammonia at a Single Molybdenum Center

Molecular nitrogen is relatively inert owing to the strength of its triple bond, nonpolarity and high ionization potential. As a result, the fixation of atmospheric nitrogen to ammonia under mild conditions has remained a challenge to chemists for more than a century. Although the Haber–Bosch process produces over 100 million tons of ammonia annually1 for the chemical industry and agriculture2, it requires high temperature and pressure, in addition to a catalyst3, to induce the combination of hydrogen (H2) and nitrogen (N2). Coordination of molecular nitrogen to transition metal complexes can activate and even rupture the strong N–N bond4 under mild conditions, with protonation yielding ammonia in stoichiometric5 and even catalytic yields6. But the assembly of N–H bonds directly from H2 and N2 remains challenging: adding H2 to a metal–N2 complex results in the formation of N2 and metal–hydrogen bonds or, in the case of one zirconium complex7, in formation of one N–H bond and a bridging hydride. Here we extend our work on zirconium complexes containing cyclopentadienyl ligands8,9 and show that adjustment of the ligands allows direct observation of N–H bond formation from N2 and H2. Subsequent warming of the complex cleaves the N–N bond at 45 °C, and continued hydrogenation at 85 °C results in complete fixation to ammonia.

Hydrogenation and cleavage of dinitrogen to ammonia with a zirconium complex

Combinations of reagents are described for the catalyzed indophenol reaction for the determination of ammonia, which produces a stable blue color. The procedure is adapted to the determination of urea after hydrolysis with urease.

http://webpages.icav.up.pt/PTDC/CVT/098487/2008/Chaney,%201962.pdf

Albert L. Chaney

Papers

Modified Reagents for Determination of Urea and Ammonia