Stanley Goldfarb

A sensitive enzymatic method for the determination of free and esterified tissue cholesterol

An enzymatic assay currently in use in clinical laboratories for quantitating total serum cholesterol has been modified for the determination of microgram quantities of tissue cholesterol. Two types of assays were developed. In the first, more generally useful method, lipids extracted with chloroform/methanol were solubilized by the addition of detergent and assayed for free and total cholesterol using laboratory prepared mixtures of reagents. Although different detergents were effective, it appears that Triton X-100 may be most universally applicable. In the second type of assay, commercially available reagent mixtures were employed for the determination of total cholesterol only. Results of both types of assays compare favorably with determination using the colorimetric assay on 3beta-hydroxysterols recovered from digitonides.

Cross-Validated Spline Methods for the Estimation of Three-Dimensional Tumor Size Distributions From Observations on Two-Dimensional Cross Sections

Abstract We study the problem of estimating the distribution of the three-dimensional radiuses of a collection of spheres, given measurements of the two-dimensional radiuses of a sample of planar cross sections. This problem arises in the estimation of the tumor size distribution of spherical microtumors induced in mouse livers following injection of a carcinogen. We first convert this problem to a form suitable for the application of cross-validated spline methods for the solution of ill-posed integral equations given noisy data. Then we develop special numerical techniques that will allow the spline methods to be accurately applied to integral equations like those associated with the present problem. We apply the resulting method to some mouse-liver data. The subject mouse liver has been completely dissected, allowing a rare comparison of the estimate with the “truth.” The statistical properties of the estimate are explored via Monte Carlo methods. The interplay between statistical and numerical analyti...

Studies of malformation syndromes of man XIB: the cerebro-hepato-renal syndrome of zellweger: Comparative pathology

A study of 11 autopsied cases of the cerebro-hepato-renal syndrome of Zellweger (ZS) is reported. All cases had severe, peristent congenital hypotonia, hepatic lobular disarray, renal cortical cysts and pulmonary hypoplasia. Many had cardiovascular malformations, hepatomegaly, cerebral cortical gyral maldevelopment and pancreatic islet hyperplasia. Additional, less frequent findings are delineated. Results of iron content studies of hepatic and renal tissues are related to age of survival and possible development of fibrosis.

Lipid synthesis and ultrastructure of adult rat hepatocytes during their first twenty-four hours in culture.

Abstract Enzymatically separated hepatocytes were cultured on collagen-coated plates and coverslips and studied during a 24 h period. Impaired lipid synthesis and ultrastructural evidence of cellular injury were apparent in freshly isolated cells immediately after perfusion. After 4–5 h in culture, most of the hepatocytes that adhered to the collagen showed marked improvement in morphology and in their ability to synthesize lipids and glycogen. Dye exclusion studies indicated that many damaged hepatocytes were selectively removed by changing the culture medium after 4 and 24 h. Evidence is also presented for the retention of feedback regulation of fatty acid and cholesterol synthesis by cultured hepatocytes. The experiments indicate that after a short recovery period, hepatocytes in primary culture are well suited for evaluating in vitro lipid synthesis. A novel thin-layer chromatographic method for determining synthesis of seven different lipid classes was also developed in the course of the study.

Sex-related differences in diurnal activities and development of hepatic microsomal 3-hydroxy-3-methyl-glutaryl coenzyme a reductase and cholesterol 7α-hydroxylase

Abstract The activities of hepatic microsomal 3-hydroxy-3-methylglutaryl CoA reductase and cholesterol 7α-hydroxylase were consistently higher (up to 3-fold) in female compared to male rats fed 2% cholestyramine for 8 h daily. In all animals studied, enzymic activities were highest 6 h after feeding began. However, 85% of the rise in cholesterol 7α-hydroxylase activity occurred in the 6 h before and 89% of the rise in 3-hydroxy-3-methylglutaryl CoA reductase activity occurred in the 6 h after feeding started. Sex-related differences in both enzymic activities first became apparent at the time of sexual maturity. Enzymic activities before weaning were generally low and a late-suckling (13–20 days) rise in cholesterol 7α-hydroxylase was not accompanied by a rise in 3-hydroxy-3-methylglutaryl CoA reductase. For all of these studies we assayed cholesterol 7α-hydroxylase at two concentrations of exogenous cholesterol to obviate problems relating to size of the cholesterol pool.

Submicrosomal localization of hepatic 3-hydroxy-3-methylglutaryl coenzyme a (HMG-CoA) reductase.

Hepatic HMG CoA reductase (mevalonate:NADP oxidoreductase (acylating CoA), EC 1.1.1.34), the rate controlling enzyme for cholesterol synthesis, is known to be located almost entirely in the microsomal fraction of the liver [ 1,2] . Two previous reports in the literature have suggested that 95% of the microsomal enzyme activity is located in the rough endoplasmic reticulum [3,4]. We wish to report the results of our own cell fractionation studies which indicate that, quite to the contrary, over 80% of the activity is found in the fractions of smooth membranes composed of smooth endoplasmic reticulum, Golgi apparatus and plasma membrane.

Erythropoietic protoporphyria: hepatic cirrhosis.

Observations on liver pathology and function were made on 12 patients with erythropoietic protoporphyria (EPP). Needle liver biopsies in 3 of 11 patients showed mild portal or periportal fibrosis without evidence of abnormal liver function. One patient (Case 12) died from hepatic cirrhosis and failure at the age of 11 years, the youngest yet reported. He was not suspected of pending liver disease until 3 months before death. The quantity of protoporphyrin present in his liver was approximately 575% of total liver weight. Fatal liver disease in EPP has occurred in 13 patients, 7 male and 6 female, in which the mean age of death was 38 years. The pathology of the liver in EPP is characteristic and is described.

Erythropoietic protoporphyria: juvenile protoporphyrin hepatopathy cirrhosis and death.

Severe hepatic cirrhosis and failure in erythropoietic protoporphyria is rare. An 11-year-old boy is described who developed protoporphyrin hepatopathy (protoporphyrin 5.75 mg/gm liver wet weight), cirrhosis, and liver failure and died.

Comparison of the adenine nucleotide translocase in hepatomas and rat liver mitochondria

Various biochemical properties of the adenine nucleotide translocase were compared with mitochondria prepared from control and host liver, and Morris hepatomas 7777, 7800 and 5123C. The transport of phosphoenolpyruvate on the adenine nucleotide translocase was found to be three to four times more active, and inhibition of the transporter by palmitoyl-CoA and atractylate considerably less in hepatoma the active transport of phosphoenolypyruvate was associated with a greater stimulation of calcium egress from the mitochondria matrix by the anion in the hepatoma. The diminished sensitivity of the adenine nucleotide translocase to palmitoyl-CoA in hepatoma mitochondria was associated with lower levels of long chain acyl-CoA esters in the whole tissue. A change in activation energy at 6 degrees C for the adenine nucleotide translocase was found in host liver mitochondria while no break point in the temperature curve was observed in hepatoma mitochondria. These results are most consistent with a change in the structure-function relationship of hepatoma mitochondria due to differences in lipid composition.

Immunohistochemical demonstration of estrophilin in mouse tissues using a biotinylated monoclonal antibody.

We have developed a direct avidin-biotin-peroxidase complex (ABC) immunohistochemical method for localization of estrophilin in mouse tissues. The method has been found especially useful for microscopic demonstration of the receptor in mouse liver, since the indirect alternative, autoradiography after injection of radiolabeled estrogens, is of no value in this organ. The ABC technique employs a biotinylated monoclonal antibody to human estrophilin (Abbot H222) which was previously shown to crossreact with the murine receptor. Cryostat-cut tissue sections which were briefly fixed were incubated with the modified antibody, and the estrophilin was revealed by subsequent exposure to ABC followed by H2O2/diaminobenzidine.