Mapping the local viscosity of non-Newtonian fluids flowing through disordered porous structures.

Flow of non-Newtonian fluids through topologically complex structures is ubiquitous in most biological, industrial and environmental settings. The interplay between local hydrodynamics and the fluid's constitutive law determines the distribution of flow paths. Consequently the spatial heterogeneity of the viscous resistance controls mass and solute transport from the micron to the meter scale. Examples range from oil recovery and groundwater engineering to drug delivery, filters and catalysts. Here we present a new methodology to map the spatial variation of the local viscosity of a non-Newtonian fluid flowing through a complex pore geometry. We use high resolution image velocimetry to determine local shear rates. Knowing the local shear rate in combination with a separate measurement of the fluid's constitutive law allows to quantitatively map the local viscosity at the pore scale. Our experimental results-which closely match with three-dimensional numerical simulations-demonstrate that the exponential decay of the longitudinal velocity distributions, previously observed for Newtonian fluids, is a function of the spatial heterogeneity of the local viscosity. This work sheds light on the relationship between hydraulic properties and the viscosity at the pore scale, which is of fundamental importance for predicting transport properties, mixing, and chemical reactions in many porous systems.

Rabbit hepatic progesterone 21-hydroxylase exhibits a bimodal distribution of activity.

Progesterone 21-hydroxylase activity varies extensively among liver microsomes prepared from individual New Zealand White (NZW) rabbits. The 21-hydroxylase activities are distributed between two groupings that differ by more than tenfold in mean activity. Both male and female animals are represented in the two groupings. However, females exhibited the higher activity more frequently than males. The 21-hydroxylation of progesterone is catalyzed by one of the liver microsomal cytochrome P-450 isozymes, form 1, and these differences in activity are suggestive of differences in the occurrence of this isozyme among NZW rabbits.

Sites of formation of the serum proteins transferrin and hemopexin.

Sites of synthesis of hemopexin and transferrin were determined by culturing various tissues of rabbits and monkeys in the presence of labeled amino acids. Labeling of the serum proteins was examined by means of autoradiographs of immunoelectrophoretic patterns as well as by precipitation in the test tubes employing immunospecific antisera. Good correlation was seen between the results obtained by the two different methods. The liver was found to be the only site of many tissues studied that synthesized hemopexin. Transferrin production was observed in the liver, submaxillary gland, lactating mammary gland, testis, and ovary.

Activation of factor XII-dependent pathways in human plasma by hematin and protoporphyrin.

Intravenous administration of hematin is effective in the treatment of acute exacerbations of the inducible porphyrias. In the course of such treatment, coagulopathies have occurred that are characterized by prolongation of prothrombin time, partial thromboplastin time, and formation of fibrin split products. In experiments in vitro with normal human plasma, we observed that hematin and protoporphyrin activated Factor XII-dependent pathways of coagulation and fibrinolysis, and that they generated kallikrein activity. Incubation of protoporphyrin with purified Factor XII resulted in activation as measured by amidolysis of a chromogenic substrate. Neither coproporphyrin, uroporphyrin, delta-aminolevulinic acid, porphobilinogen, or bilirubin activated Factor XII-dependent pathways. Exposure of serum containing added uroporphyrin, coproporphyrin, and protoporphyrin, but not hematin, to ultraviolet light (405 nm) resulted in activation of the classical pathway of the complement system. On the other hand, exposure of plasma containing uroporphyrin or coproporphyrin to ultraviolet light did not result in activation of Factor XII-dependent pathways.

Effect of microsomal cytochrome P-450 isozyme induction on the mutagenic activation of 2-aminoanthracene.

Two rabbit microsomal cytochrome P-450 isozymes. Forms 4 and 6, which are differentially induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the liver in an age-dependent fashion, catalyze the activation of 2-aminoanthracene to a mutagen in the Ames Salmonella mutagenesis assay. We have shown previously that in the presence of saturating concentrations of 2-aminoanthracene, Form 4 is 15-fold more active than Form 6 in the activation of this mutagen. Similar differences in the activation of 2-aminoanthracene are observed between liver microsomes isolated from TCDD-treated adult rabbits, in which Form 4 predominates, and microsomes from rabbit neonates exposed transplacentally to TCDD prior to birth, in which Form 6 predominates. However, when the extent of mutagenesis is limited by the amount of 2-aminoanthracene and is independent of the rate of activation, the number of revertants produced is similar for microsomes isolated from either newborn or adult TCDD-treated rabbits. Under these conditions, the extent of mutagenesis obtained for a given amount of 2-aminoanthracene will depend on the balance between activation and competing reaction pathways leading to detoxication. Thus, differences in the rate of activation between adult and newborn microsomes are probably offset by similar differences in the rates of competing pathways of metabolism. This is consistent with the finding that the overall rate of 2-aminoanthracene metabolism by the adult microsomes is greater than that of the neonate. In order for the extent of mutagenesis to be independent of rate, the half-life of 2-aminoanthracene was seen to be less than approximately 12 min.

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin and phenobarbital on the occurrence and distribution of four cytochrome P-450 isozymes in rabbit kidney, lung, and liver.

The effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and of phenobarbital (PB) on the distribution and occurrence of four cytochrome P-450 isozymes, Forms 2, 3, 4, and 6, in the kidney, lung, and liver of adult male rabbits was investigated using immunofluorescence. In the kidney, Forms 2 and 3 were localized in the proximal tubules of both untreated and PB-treated animals, while antibodies to Forms 4 and 6 showed weak to negative staining. In TCDD-pretreated animals, Forms 4 and 6 appeared in the renal endothelium, in addition to staining the proximal tubular epithelium intensely. Form 2 was the only isoenzyme of those studied found to be present in the lungs of normal and PB-pretreated rabbits; it was also present in lungs of TCDD-pretreated rabbits. Form 3 was not detected in any of the rabbit lungs examined. Forms 4 and 6, while not apparent in the lungs of normal or PB-treated animals, were found in the lungs of TCDD-treated animals and also appeared in the endothelium of the pulmonary arteries and veins. All forms tested were present in control liver. The staining for Form 2 was intense in the livers of PB-pretreated animals, as was the staining for Forms 4 and 6 in the livers of TCDD-pretreated animals. Our results indicate that, while PB altered the intensity of staining for Form 2 in the liver and kidney, TCDD altered both the staining intensity and distribution of the isozymes in kidney, lung, and liver, producing, for example, a localization of Forms 4 and 6 in the endothelium of both the kidney and lung which was not seen in either untreated or PB-pretreated rabbits.

Catalysis of divergent pathways of 2-acetylaminofluorene metabolism by multiple forms of cytochrome P-450.

Four highly purified forms of rabbit hepatic, microsomal cytochrome P-450 catalyze the N- and ring-hydroxylation of 2-acetylaminofluorene (AAF) at different rates. Form 4, the major form of the cytochrome induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in adult rabbit liver, catalyzed the N-hydroxylation of AAF more rapidly than did the other three forms. N-Hydroxy-2-acetylaminofluorene accounted for 70% of the metabolites formed by the action of this cytochrome. Form 6, the major form of the cytochrome induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in neonate rabbit liver, and Form 3, a constitutive form of the cytochrome, both metabolized AAF at one-half the rate observed for Form 5. Phenols accounted for more than 90% of the metabolites produced by these two cytochromes. The major phenobarbital-inducible cytochrome P-450, Form 2, exhibited practically no catalytic activity (< 1% of the other forms) with AAF as a substrate. Since N- and ring-hydroxylation are thought to represent divergent pathways of carcinogen metabolism (activation versus detoxification), the differential occurrence of the various cytochrome forms should affect the balance between these two reaction pathways. In this sense, cytochrome P-450 induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin differentially affects the magnitude and direction of in vitro microsomal metabolism of AAF as a function of age.

Studies on the efflux of heme from biological membranes.

It is unknown how heme is distributed intracellularly from its site of synthesis in the mitochondria to other organelles. In previous work (Biochemistry 23, 3715, 1984) the transfer of heme from lipid bilayers to soluble proteins had been found to be independent of the recipient proteins' affinity for heme. Here, we investigated whether proteins are involved in the transfer of heme from biological membranes into aqueous media. We followed the release of 14C-labeled heme, from mitochondria preloaded with the heme, to BSA and found that only about 28%, of the heme was extracted on the first wash. After the third wash 35-50% of the heme that had been partitioned into the membranes was extracted. Fourth and fifth washes with BSA or a cytosolic heme-binding protein (HBP, also known as liver fatty acid binding protein) removed only insignificant amounts of 14C-labeled heme. Similarly, a large portion of the preloaded 14C-labeled heme could not be extracted from a variety of isolated membranes (inner and outer mitochondrial membranes, plasma membranes of liver cells, kidney cortex cells and erythrocyte membranes). By contrast, essentially all [14C]palmitate preloaded in biological membranes and all 14C-labeled heme preloaded in synthetic membranes was released to albumin (Biochemistry 23, 3715, 1984). These observations suggest that, in general, heme associates with membrane components which can be distinguished into two compartments. One compartment releases its heme spontaneously, while another compartment binds heme so tightly that a specific process has to be evoked for its release.

Effect of porphyrinogenic agents on protein synthesis and bilirubin formation by the isolated perfused rat liver.

We established an isolated rat liver perfusion system for the study of heme catabolism. The liver of rats fasted for 48 h is perfused with an erythrocyte-free medium. Ultrastructural analysis shows integrity of all subcellular organelles with the exception of minor alterations in the rough endoplasmic reticulum. The perfused liver synthesizes serum proteins at a constant rate for 5 h. Albumin is secreted at a mean rate of 17 +/- 2 mg/h per 100 g liver, hemopexin at 5.0 +/- 0.7, haptoglobin at 3.2 +/- 0.6 and transferrin at 5.1 +/- 0.8 mg/h per 100 g liver. The mean ratio of ATP : ADP is 3.5 +/- 0.1, and that of lactate: pyruvate 27 +/- 6. The rate of conversion of heme into bilirubin is comparable to that reported for in vivo studies. A minimal effect on protein synthesis is observed after administration of the porphyrinogenic agents, allylisopropylacetamide (AIA) and 3,5-diethoxycarbonyl-1,4 dihydrocollidine (DDC). Pretreatment of the rats with the iron chelator, Desferal, causes a 3-4-fold increase in hemopexin but not in albumin and transferrin synthesis. A striking 2-3-fold enhancement of bile bilirubin production follows treatment with DDC and Desferal, but not with AIA. The amount of bilirubin formed from heme added to the perfusate is reduced by AIA and DDC and enhanced by Desferal treatment. It is proposed that unavailability of iron in a certain hepatic tissue pool causes protoporphyrin IX accumulation which may serve as an alternate source for bilirubin production.

The aromatic and heme chromophores of rabbit hemopexin. Difference absorption and fluorescence spectra.

Spectrophotometric and fluorimetric techniques were employed to charcterize the environment of the heme chromophore of rabbit hemopexin and to monitor changes in the environment of aromatic amino acid residues induced by the interaction of hemopexin with porphyrins and metalloporphyrins. Difference spectra showed maxima at 292 and 285 nm when hemopexin binds heme or deuteroheme but not deuteroporphyrin. These maxima are attributed to alterations in the local environment of tryptophan and tyrosine residues. Spectro-photometric titrations of the tyrosine residues of hemopexin, heme-hemopexin and hemopexin in 8 M urea showed apparent pK values at 11.4, 11.7, and 10.9 respectively. Perturbation difference spectra produced by 20% v/v ethylene glycol are consistent with the exposure of 6-8 of the 14 tyrosine residues and 6-8 of the 15 tryptophan residues of rabbit hemopexin to this perturbant. Only small differences were found between the perturbation spectra of apo- and heme-hemopexin near 290 nm, suggesting that slight or compensating changes in the exposure to solvent of tryptophan chromophores occur. In the Soret spectral region, the exposure of heme in the heme-hemopexin complex to ethylene glycol was 0.7, relative to the fully exposed heme peptide of cytochrome c. The fluorescence quantum yields of rabbit apo- and heme-hemopexin were estimated to be 0.06 and 0.03, respectively, compared to a yield of 0.13 for L-tryptophan. Iodide quenched 50% of the fluorescence of the deuteroheme-hemopexin complex. Cesium was not an effective quencher. Modification of approximately, 4 tryptophan residues with N-bromosuccinimide also decreased the relative fluorescence of apo-hemopexin by 50% and concomitantly reduced the heme-binding ability of the protein by 70%. The existence of sterically unhindered tryptophan residues in either apo- heme-hemopexin is unlikely since no charge transfer compelxes between these proteins and N-methylnicotinamide were detected.

Interaction of rabbit hemoplexin with copro- and uroporphyrins.

Rabbit hemopexin forms equimolar complexes in vitro with the I and III isomers of both coproporphyrin and uroporphyrin. The apparent dissociation constants (Kd) of these complexes are estimated to be 4-10(-7) M for coproporphyrin-hemopexin and 10(-6) M for uroporphyrin-hemopexin by equilibrium dialysis and quenching of protein fluorescence. Results of competitive binding experiments suggest that all four porphyrins bind at the heme-binding site of hemopexin, and that the relative affinity of rabbit hemopexin for these porphyrins is: deuteroheme greater than coproporphyrin I or III greater than uroporphyrin I or III. These findings provide further evidence that hemopexin may function as a transport protein for circulating coproporphyrins as well as for heme.

Long-term intercalation of residual hemin in erythrocyte membranes distorts the cell.

The effect of long-term incubation of residual globin-free hemin on whole red blood cell and isolated cytoskeletal proteins was studied. Hemin at concentrations found in pathological red cells was inserted to fresh erythrocytes. Increased hemolysis developed in the hemin-containing cells after a few days at 37 degrees C and after about four weeks at 4 degrees C. Since lipid and hemoglobin peroxidation did not depend on the presence of hemin, time-dependent effects on the cytoskeleton proteins were studied. Observations were: (1) spectrin and protein 4.1 exhibited a time-dependent increasing tendency to undergo hemin-induced peroxidative crosslinking. (2) The ability of the serum proteins, albumin and hemopexin, to draw hemin from spectrin, actin and protein 4.1 decreased with time of incubation with hemin. These results were attributed to time-dependent hemin-induced denaturation of the cytoskeletal proteins. Albumin taken as a control for physiological hemin trap was unaffected by hemin. Small amounts of hemo-spectrin (2-5%) were analyzed in circulating normal cells, and this in vivo hemo-spectrin also failed to release hemin. It was concluded that slow accumulation of hemin, a phenomenon increased in pathological cells, is a toxic event causing erythrocyte destruction.

Interaction of rabbit hemopexin with bilirubin.

The interaction of hemopexin with bilirubin was characterized by spectrophotometric, fluorimetric and circular dichroic techniques. Hemopexin rapidly forms an equimolar complex with libirubin that has an apparent dissociation constant Kd, of 7.5.10(-7) M. The association alters the absorption band of bilirubin near 150 nm, quenches the fluorescence of tryptophan residues of hemopexin, enhances the fluorescence of bilirubin, and induces strong ellipticity extrema in bilirubin of --60 . 10(3) deg . cm2 . dmol-1 at 465 nm and +70 . 10(3) deg . cm2 . dmol-1 at 415 nm. However, the conformation-sensitive ellipticity aband at 231 nm of hemopexin is not altered. In displacement experiments using circular dichroism, heme readily replaced bound bilirubin, indicating that bilirubin and heme are bound at the same site on hemopexin. Even at molar ratios of hemopexin to albumin of 3 to 1, human serum albumin removes bilirubin from hemopexin. Hemopexin is thus unlikely to have a role in the transport of bilirubin in serum.

Proton NMR study of the heme complex of hemopexin.

Proton nuclear magnetic resonance spectroscopy of the complex of heme with hemopexin, a plasma protein with an exceptionally high affinity for heme, is reported. Characteristic spectra are shown for heme.hemopexin of cow, human, rabbit, and rat. Each of these spectra demonstrate that the iron of heme bound by hemopexin is paramagnetic and low-spin. Rabbit heme.hemopexin, which exhibits the best signal-to-noise ratio, is studied in detail. Deuterium isotope labeling experiments indicate that the methyls in heme positions 1-, 3-, and 8- are resolved downfield from the protein envelope of resonances; the 5-methyl may lie in the -5 to +12 ppm region. Two-dimensional nuclear Overhauser effect spectroscopy locates other protons of the heme periphery, including from the 2-vinyl. Strongly relaxed upfield resonances are identified and assigned to protons on the axial ligands. Cyanide interaction with heme.hemopexin produces an additional low-spin adduct.

Effects of hexachlorobenzene and iron loading on rat liver mitochondria.

The effects of hexachlorobenzene treatment and simultaneous iron-overload on the iron and porphyrin content of rat liver and rat liver mitochondria have been examined. In order to assess damages to the mitochondrial membrane occurring with these treatments, the content of malondialdehyde and selected functional properties of mitochondria were compared with those from control animals. Prolonged intake of hexachlorobenzene (8 weeks) resulted in a strikingly increased level of porphyrins together with a moderate increase in iron concentration. Simultaneous administration of hexachlorobenzene and iron-dextran caused the porphyrin level to reach 25% of the amount induced by hexachlorobenzene alone. The iron concentrations in liver as well as in liver mitochondria are also decreased under these conditions, as compared to the effect of iron-dextran. In contrast, the effects of hexachlorobenzene combined with iron-dextran on mitochondrial oxidative phosphorylation and malondialdehyde content are greater than those of either hexachlorobenzene or iron-dextran. These data suggest that porphyrin accumulation per se causes little deleterious effect and that both agents administered together act synergistically in causing damage to the mitochondrial membrane.

Griseofulvin causing hyperhemopexinemia and hepatic proliferation in mice: an in vivo and in vitro study.

Concentrations of hemopexin, a porphyrin-binding serum protein synthesized exclusively in the liver, increased significantly and concomitantly with levels of erythrocyte and liver protoporphyrin and coproporphyrin in mice made porphyric with 1% griseofulvin in the feed. Liver weights of porphyric mice increased remarkably. In comparison with controls, the ratio of the weight of normal to porphyric livers was at 10 days 1:2.1, at 21 days 1:2.8, and at 46 days 1:3.8. This increase in liver size was accompanied by increased cell division. The hepatic hyperplastic tissue fragments survived in vitro for several weeks and could be subcultured. The cultured cells, like those of the original liver, showed intense protoporphyrin fluorescence in the cytoplasm.

Serum proteins as mediators of hemin efflux from red cell membranes: specificity of hemopexin.

The involvement of the serum heme-binding proteins hemopexin and albumin in the clearance of erythrocyte membranes from toxic hemin was compared. In the presence of hemopexin initial rates of hemin efflux from resealed ghosts were faster and the amount of extracted hemin larger. When hemin-containing ghosts were treated with a protein mixture of 1:45 hemopexin to albumin, as present in serum, most of the hemin was extracted in the form of heme-hemopexin. It was concluded that hemopexin is the serum protein responsible for heme extraction from cell membranes.

Hyaluronan-binding properties of human serum hemopexin.

Hemopexin, the heme-binding serum glycoprotein, exhibits a complex electrophoretic pattern on two-dimensional immunoelectrophoresis on agarose gels into which hyaluronic acid is incorporated in the first and monospecific anti-hemopexin in the second dimension. This heterogeneity reflects a range of interactions of hemopexin isoforms with hyaluronic acid. Electrophoretic patterns of individual human sera greatly differ in their contents of hyaluronan-interacting hemopexin species. Hemopexin itself has no hyaluronidase activity.

A turbidometric assay for measuring proteins in culture media.

An immunoturbidometric assay was developed for the measurement of proteins in culture fluids of hepatocytes. The assay is simple to perform and avoids the biohazards associated with radioimmunoassays. The limit of detection of this assay exemplified by hemopexin and transferrin is 5 ng/ml protein. This degree of sensitivity is attained by incorporating into the procedure the addition of polyethylene glycol to enhance formation of primary antigen-antibody complexes and of a second antibody to further increase the immune complex size, which favors the ratio of specific to background light scattering.

Modulation of hepatic heme-binding Z protein in mice by the porphyrogenic carcinogens griseofulvin and hexachlorobenzene.

Some reports link human hepatic porphyria with a risk of hepatocellular carcinoma. Hepatic protoporphyria and uroporphyria were induced in mice by feeding griseofulvin and hexachlorobenzene (HCB), respectively. These chemicals also cause liver cancer. Hepatic immunoreactive cytosolic levels of heme-binding Z protein (HBP) were reduced by 81% (griseofulvin) and 55% (HCB). In contrast, both treatments caused a greater than 4-fold increase in the immunoreactive levels of glutathione S-transferase isozymes (GST) which like HBP also bind heme. Unlike in vitro studies in the presence of porphyrins, no cross-linking of HBP was observed in vivo.