Multiple-indicator dilution studies of labeled bilirubin uptake were carried out on isolated perfused rat livers with variable ligandin concentrations (from normal and thyroidectomized animals with and without phenobarbital pretreatment). Ligandin concentrations, measured immunologically, increased 25% after thyroidectomy and approximately doubled after phenobarbital pretreatment but decreased to normal during perfusion in the thyroidectomized nonpretreated group. A distributed two-compartment model was fitted to the dilution data and estimates of influx, efflux, and sequestration coefficients were obtained. Influx and sequestration coefficients did not vary significantly between the groups. Efflux coefficients were significantly smaller (P less than 0.001), and hepatic ligandin concentrations were significantly larger (p less than 0.001) in phenobarbital-treated rats than in other groups. The efflux coefficient varied inversely with ligandin concentration and the volume of distribution in tissue, as perceived from the plasma space, increased in proportion to the concentration of ligandin. The increased net uptake of tracer bilirubin by the liver of phenobarbital-pretreated animals is due to decreased tracer efflux secondary to the increase in intracellular binding of bilirubin by ligandin.

No abstract

The multiple-indicator dilution technique was utilized to examine the hepatic uptake of albumin-bound labeled palmitate from the portal vein blood of the pentobarbital-anesthetized dog, in a fasted state and after infusion of a variety of compounds that were expected to bind to Z protein, the cellular cytosolic protein binding free fatty acids, and their acyl-CoA derivatives. Analysis of the data indicates that after infusion of alpha-bromopalmitate, 16-bromo-9-hexadecenoate, and sulfobromophthalein sodium (which also bind to albumin), the palmitate label influx, efflux, and metabolic sequestration (removal of label from the pool of free fatty acids able to leave the cell) all increase and that, after infusion of flavaspidic acid, label efflux and metabolic sequestration increase. In vitro competitive binding studies carried out on the cellular cytosol indicat that the basis for the increase in efflux and metabolic sequestration is displacement of labeled palmitate from high affinity sites on the intracellular Z protein (which are presumably in equilibrium with and may be taken to be representative of other intracellular binding sites). These studies also suggest that increased uptake is due to similar displacement from high affinity sites on serum albumin.

In the present study we describe a simple and fast method to measure the concentration of total free amino acids in very small amounts of biological tissues. The procedure described here is based on the reaction of free amino acids with o-phthaldialdehyde (OPA) in the presence of a reducing agent, beta-mercaptoethanol (MET), to give a complex which can be measured by fluorescence. It is a very rapid process and has the same reliability as the conventional ninhydrin method of Moore and Stein but is about 500 times more sensitive. The sensitivity of the new protocol is such to permit the determination with high reliability of very small amounts of free amino acids at picomole levels, either in a standard amino acid mixture or in biological tissues, without chromatographic separation of the amino acids. It is particularly useful when the amount of the sample is very low, e.g. on a single pituitary or pineal gland of small animals or on single cells, such as oocytes or eggs, as well as single ganglions or axons of marine invertebrates.

Serum-free, hormonally defined media have been developed for optimal growth of a rat hepatoma cell line. The cells' hormonal requirements for growth are dramatically altered both qualitatively and quantitatively by whether they were plated onto tissue culture plastic or collagenous substrata. On collagenous substrata, the cells required insulin, glucagon, growth hormone, prolactin, and linoleic acid (bound to BSA), and zinc, copper, and selenium. For growth on tissue culture plastic, the cells required the above factors at higher concentrations plus several additional factors: transferrin, hydrocortisone, and triiodothyronine.To ascertain the relative influence of hormones versus substratum on the growth and differentiation of rat hepatoma cells, various parameters of growth and of liver-specific and housekeeping functions were compared in cells grown in serum-free, hormonally supplemented, or serum-supplemented medium and on either tissue culture plastic or type I collagen gels. The substratum was found to be the primary determinant of attachment and survival of the cells. Even in serum-free media, the cells showed attachment and survival efficiencies of 40-50% at low seeding densities and even higher efficiencies at high seeding densities when the cells were plated onto collagenous substrata. However, optimal attachment and survival efficiencies of the cells on collagenous substrata still required either serum or hormonal supplements. On tissue culture plastic, there was no survival of the cells at any seeding density without either serum or hormonal supplements added to the medium. A defined medium designed for cells plated on tissue culture plastic, containing increased levels of hormones plus additional factors over those in the defined medium designed for cells on collagenous substrata, was found to permit attachment and survival of the cells plated into serum-free medium and onto tissue culture plastic.Growth of the cells was influenced by both substrata and hormones. When plated onto collagen gel substrata as compared with tissue culture plastic, the cells required fewer hormones and growth factors in the serum-free, hormone-supplemented media to achieve optimal growth rates. Growth rates of the cells at low and high seeding densities were equivalent in the hormonally and serum-supplemented media as long as comparisons were made on the same substratum and the hormonally supplemented medium used was the one designed for that substratum. For a given medium, either serum or hormonally supplemented, the saturation densities were highest for tissue culture plastic as compared with collagen gels.Differentiative functions, as measured by various enzymatic assays and by analyses of RNA populations for liver-specific and housekeeping mRNA, were also influenced by synergies between hormones and substratum. Some known liver-specific functions, such as albumin,