Sidney S. Levin

PHOTOCHEMICAL ACTION SPECTRUM OF THE TERMINAL OXIDASE OF MIXED FUNCTION OXIDASE SYSTEMS.

The reversal of the carbon monoxide inhibition by bands of monochromatic light was determined for the oxidative demethylation of codeine and monomethyl-4-aminopyrine and the hydroxylation of acetanilide by rat liver microsomes and for the hydroxylation of 17-hydroxyprogesterone at carbon-21 by bovine adrenocortical microsomes. Maximum reversal occurred at 450 millimicrons, the light absorption maximum of the CO compound of the CO-binding pigment of microsomes. The agreement between photochemical action spectrum and spectrophotometric difference spectrum supports the conclusion that the CO-binding pigment is the terminal oxidase of mixed function oxidase systems of mammals.

Analysis of Saturated and Unsaturated Phospholipids in Biological Fluids

Abstract Cupric acetate (3% in 8% phosphoric acid) as a charring agent reacts only with unsaturated phospholipids while cupric sulfate (10% in 8% phosphoric acid) reacts with both saturated and unsaturated phospholipids. Thus, the amount of saturated phospholipid in a zone on a thin layer chromatogram (TLC) can be calculated by the difference in reactivity. An evaluation of methods shows that direct application of biological samples to TLC for separation and quantitation of phospholipids is reproducible. The use of these techniques for a number of different samples is described.

Evidence for diplasmalogen as the major component of rabbit sperm phosphatidylethanolamine

The question of whether diplasmalogens [1,2-di(O-1′-alkenyl) phosphatidyl derivatives] make up part of the plasmalogen component of cell phospholipids was examined using rabbit epididymal spermatozoa. These cells are readily obtained as a highly homogeneous suspension and long have been known to have high plasmalogen content. Phospholipids were determined by thin layer chromatography (TLC) with CuSO4 staining. Plasmalogens were determined by hydrolysis of the phospholipids with TCA/HCl, followed by TLC and CuSO4 staining. Ethanolamine derivatives were determined by ninhydrin. The phosphatidylethanolamine (PE) content of these cells was 29±2 μg/108 cells, 90% of which was assayed as diplasmalogen and 10% as diacyl PE. No monoplasmalogen could be detected. The presence of diplasmalogen as the major component of PE was given further support from infrared and proton nuclear magnetic resonance (1H-NMR) spectroscopy, which showed the presence of O-1′-alkenyl substituents but near absence of O-acyl substituents. The phosphatidylcholine (PC) content of the cells was 104±5 μ/108 cells, of which 50% was monoplasmalogen with the 1′-alkenyl group on the 2 position of the glycerol moiety. No diplasmalogen was found in PC. The other phospholipids in rabbit sperm were phosphatidylglycerol (PG), cardiolipin (CL), sphingomyelin (SP) and lysophosphatidylcholine (LPC). Phosphatidylserine (PS) and phosphatidylinositol (PI) were present at the limits of detectability of the TLC method. None of these phospholipids contained plasmalogen. The PE component of rabbit sperm phospholipids appears to differ from that of the other cells in having the previously unreported diplasmalogen as its major constituent.

Separation of conjugated bile acids by reverse phase thin layer chromatography

Seven conjugated bile acids were separated by 1-dimensional reverse phase thin layer chromatography using ethanol/0.3 M calcium chloride/dimethylsulfoxide (25∶25∶2) as mobile phase.

Analysis of Plasmalogens by In Situ Reaction on Thin Layer Chromatograms

Abstract An “in situ” hydrolysis method that attacks only the enol ether double bond of plasmalogen is described. Alkyl and unsaturated phosphollpids are not affected. Treatment of the plasmalogen on thin layers of silica gel with solutions of trichloroacetic acid in dilute hydrochloric acid result in more complete hydrolysis of the vinyl-ether linkage. After development of the chromatogram differential quantitation using copper sulfate charring (1) and densitometry will give the amount of vinyl-ether lipid present in the sample. The results of the method with ethanolamine plasmalogen are presented.

STUDIES ON THE PARTIALLY PURIFIED HEME PROTEIN P‐450 FROM THE ADRENAL CORTEX*

The reconstituted steroid 1 lp-hydroxylase is a particularly suitable system for studying the individual reactions involved in hydroxylations catalyzed by mixedfunction oxidase. This system is that described elsewhere by our laboratory and is composed of a specific flavoprotein (Fp), an iron-sulfur protein (ISP), and a partially purified heme protein P-450 preparation.’ The electron transport scheme and the oxidation-reduction properties found in our previous work are outlined in FIGURE 1. These studies furthermore have demonstrated that TPNH and the flavoprotein are dispensable and that the electrons for 11/3-hydroxylation can be supplied by reduced ISP.’*8 In this paper we wish to describe in some detail the results of recent studies on the properties of the individual components of this system.

The metabolism and excretion of enzyme-inducing doses of phenobarbital by rats with bile fistulas

The metabolism of enzyme-inducing doses of 14C-phenobarbital injected i. p. into bile duct-cannulated rats has been studied using improved chromatographic separation and quantification techniques. In animals with bile fistulas most of the 14C-phenobarbital was excreted in bile as p-hydroxyphenobarbital conjugated with glucuronic acid. In urine the main substance found was phenobarbital, with significant amounts of p-hydroxyphenobarbital and varying amounts of its glucuronide conjugate. Animals without bile fistulas excreted 80% dose of phenobarbital in the urine; metabolites were free phenobarbital, p-hydroxyphenobarbital and conjugated material. Approx 90% of the conjugated material was the glucuronide. Only free phenobarbital and p-hydroxyphenobarbital were found in the faeces. Animals drinking plain water excreted 50-65% dose of phenobarbital (80 mg/kg) in bile and the remainder mainly in the urine, whereas superhydrated animals (drinking 5% glucose and 0.9% NaCl) excreted 90% of the dose as free phenobarbital in the urine. Phenobarbital is the only labelled material detectable in hepatic tissue and portal, vena caval or aortic blood, which indicates that phenobarbital is the enzyme-inducing substance and that liver and kidney rapidly eliminate all metabolites. Metabolism of phenobarbital in vivo is a complex process involving interaction of hepatic and intestinal metabolism, partial readsorption from the intestinal tract and renal elimination.

Sample Application in Thin Layer Chromatography

Abstract Resolution in thin layer chromatography depends on the ability to properly apply the sample to the origin. Thin lines or streaks are recommended. Preabsorbent layers facilitate sample application since thin lines result. Automatic sample applicators are prerequisite if preabsorbent layers are not used and more than 15 μ1 of the solution is to be applied.

Biophysical and catalytic properties of the phenobarbital p-hydroxylase—a cytochrome P-450 dependent mixed function oxidase

1. A sensitive method to detect and quantify the products of phenobarbital (PB) hydroxylation by model chemical systems and by biological systems has been developed. 2. Chemical model systems hydroxylate PB in the p-position of the phenyl ring and form one or two additional oxidation products, while in vitro and in vivo (bile fistula rats) biological systems hydroxylate PB only in the p-position. 3. Phenobarbital hydroxylation rates in vitro are of the order of 0.007 nmol/nmol cytochrome P-450 per min. These values are decreased by pretreatment of the rats with inducing doses of phenobarbital. 4. Enzymes catalysing the p-hydroxylation reaction of phenobarbital are localized in the microsomes and have the biophysical and chemical properties that are usually associated with cytochrome P-450-dependent mixed function oxidases.

Characterization of Phosphoglycerides by Chemical and Enzymatic Hydrolysis on Thin Layer Plates In Situ

Abstract The use of chemical and enzymatic reactions in situ on thin layer chromatograms for characterization of phosphoglycerides is described. Acid and alkaline hydrolysis under specific conditions can differentiate acyl, alkyl and alkenyl moieties. Phospholipases A2, C and D were shown to give complete and specific hydrolysis of phospholipids directly on the chromatograms.