where subscripts e and c refer to extracellular and cytosolic spaces, respectively, several lines of evidence support the assumption that near-equilibrium conditions exist for processes 1 through 3 during the normoxic steady state of rat liver [l-31.Since 1965, in studies with the isolated hemoglobin free perfused organ [4,5], extensive use was made of this system in the direction from right to left, using the ratio interest was focussed on the other direction in order to obtain detailed information on the NADH system of liver oytosol which was previously not accessible. Extracellular [lactate]/[pyruvate] was used as experimental variable, and dependent changes in cytosolic NADH were observed by a variety of methods, in particular by continuous measurement of fluorescence and absorbance of perfused liver a t pyridine-nucleotide-specific wavelengths.By means of mathematical extrapolation of titration results in a double reciprocal plot derived on the basis of the mass action law, the maximal

A patient with a cerebro-hepato-renal syndrome was investigated. The visceral manifestations were those of the Zellweger syndrome (ZS); however, the child exhibited muscular hypertonia and survived into the 2nd year of life. Ultramicroscopically, hepatocytes were lacking peroxisomes, but, contrary to findings in one patient with ZS [2], contained smooth endoplasmic reticulum. No catalase was found by histochemistry or spectroscopy. Mitochondria showed normal succinate and glutamate respiration, and normal coupling of respiration to the phosphorylation potential. The cytochrome (cyt) content was diminished to one-third with an abnormally inversed redox pattern of the respiratory chain in the controlled state, cyt b being 5%, cyt c 23% reduced. The oxygen affinity of cyt a3 was normal. These findings exclude a defect in the nonheme iron protein region of the respiratory chain as described in ZS [2], but point to a functional abnormality of cyt b in out patient.

1. The interactions of a 'Type I' compound, hexobarbital, and of a 'Type 11' compound, metyrapone, with the mixed function oxidase were demonstrated by spectrophotometric techniques in hemoglobin-free perfused livers from phenobarbital-treated rats. The binding signal for hexobarbital occurred when cytochrome P-450 was mainly oxidized (normoxic perfusion) or reduced (anoxic perfusion), The binding signal disappeared as hexobarbital was metabolized.2. Functional aspects were demonstrated by simultaneous readings of (a) the reduced cytochrome P-450-CO complex, providing an indication of the degree of reduction of cytochrome P-450; (b) surface fluorescence (366 --f >420 nm); and (c) oxygen uptake.The steady state level of reduced P-450-CO was increased when substrate was metabolized. Surface fluorescence intensity decreased following hexobarbital addition. This decrease was explained partly by a decrease of the tissue level of NADPH, and partly also by interference of the P-450-hexobarbital complex a t the excitation wavelength. Extra oxygen uptake was 2.2-2.4 moles per mole of hexobarbital. Half-maximal concentration of hexobarbital was 59 and 75 pM for extra oxygen uptake and binding to the oxidase, respectively, in agreement with data for enzymatic activity and binding from isolated liver microsomes.The parameters (a, b, c) returned to their original level as hexobarbital was metabolized, similar to findings with the steroid 21 -hydroxylase system of isolated adrenocortical microsomes.3. The overall degree of reduction of the NADPH system, NADPH/NADPH+NADP+, decreased from 0.8 to 0.7 in livers from phenobarbital-treated rats when hexobarbital was added. I n contrast, the free cytosolic NADH system did not undergo redox changes as indicated by a constant ratio of lactatelpyruvate in the perfusate.4. Mitochondria1 NADH dehydrogenase was shielded against the barbiturate by the endoplasmic reticulum as a consequence of phenobarbital induction.A wealth of information about the hepatic mixed function oxidase system has accumulated during recent years from studies with isolated microsomal fractions [1, 1 a]. Direct insight into this system within the intact organ is possible by spectrophotometric and fluorometric techniques in the hemoglobin-free perfused rat liver [2,3]. I n the present study, simultaneous measurement of the effect of external substrate on four parameters is described, furthering the understanding of this system when , integrated in the dynamics of the cell: (a) spectral interaction of external substrate with cytochrome P-450, the terminal oxidase ; secondly, the steady state level of the reduced P-450-CO complex, both Enzyme. Mixed function oxidase (EC 1.99.1.1.). 35.measured by spectrophotometry of perfused liver in the transmitted light; (b) effect of external substrate on reduced pyridine nucleotide surface fluorescence; and (c) oxygen uptake, serving as a measure of the rate of mixed function oxidation.The interrelations between mixed function oxidase and other cellular redox systems such as the ...

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