Development of monoclonal antibodies to the malondialdehyde-deoxyguanosine adduct, pyrimidopurinone.

Malondialdehyde (MDA), an endogenous product of lipid peroxidation and prostaglandin biosynthesis, is mutagenic in bacterial and mammalian cells and carcinogenic in rats. In order to determine whether MDA-modified bases are formed in nucleic acids in vivo, sensitive immunoassays to detect MDA-DNA and MDA-RNA adducts are being developed in our laboratory. Murine monoclonal antibodies reactive with the MDA-deoxyguanosine adduct 3-beta-D-erythro-pentofuranosylpyrimido[1,2-alpha]purin-10(3H)-one (M1G-R) were prepared and characterized. Several MDA-modified nucleosides and deoxynucleosides and structural analogs were synthesized and characterized and were compared as competitive inhibitors in enzyme-linked immunosorbent assays (ELISAs). Less than 5 fmol of M1G in MDA-modified DNA was detected in a direct ELISA, and antibody binding to the modified DNA was competitively inhibited by free M1G-dR. DNA from Salmonella typhimurium treated with concentrations of MDA that induce reversion to histidine prototrophy was enzymatically digested, and M1G-dR was quantitated by competitive ELISA. Over a range of MDA concentrations from 10 to 40 mM, the level of M1G residues in bacterial DNA increased from 0.2 to 2.5/10(6) base pairs.

Analysis of the malondialdehyde-2'-deoxyguanosine adduct pyrimidopurinone in human leukocyte DNA by gas chromatography/electron capture/negative chemical ionization/mass spectrometry.

A method is described for the assay of the major malondialdehyde-deoxyguanosine adduct (M1G) based on immunoaffinity purification and gas chromatography/electron capture/negative chemical ionization/mass spectrometry. A stable isotope of M1G-deoxyribose ([2H2]M1G-dR) was used as an internal standard. Recovery of internal standard throughout the entire assay procedure was approximately 40%. The assay showed a linear response over a range of 10-1000 pg of M1G-dR and was verified by analysis of a synthetic. M1G-containing oligomer. The limit of detection in biological samples was 100 fmol/sample, corresponding to 3 adducts/10(8) bases for 1 mg of DNA. DNA was isolated from the blood of 10 healthy human donors, and M1G levels were measured. A mean value of 6.2 +/- 1.2 adducts/10(8) bases was obtained, with no obvious differences bases on age or cigarette smoking. A small, but statistically significant difference was observed between the levels in females (5.1 +/- 0.4 adducts/10(8) bases) and males 6.7 +/- 1.1 adducts/10(8) bases). The presence of M1G in leukocyte DNA was further verified by analysis using liquid chromatography/electrospray ionization mass spectrometry.

Detection of endogenous malondialdehyde-deoxyguanosine adducts in human liver.

Endogenous DNA adducts may contribute to the etiology of human genetic disease and cancer. One potential source of endogenous DNA adducts is lipid peroxidation, which generates mutagenic carbonyl compounds such as malondialdehyde. A sensitive mass spectrometric method permitted detection and quantitation of the major malondialdehyde-DNA adduct, a pyrimidopurinone derived from deoxyguanosine. DNA from disease-free human liver was found to contain 5400 adducts per cell, a frequency comparable to that of adducts formed by exogenous carcinogens.

Analysis of the malondialdehyde-2'-deoxyguanosine adduct in rat liver DNA by gas chromatography/electron capture negative chemical ionization mass spectrometry.

Malondialdehyde (MDA), a product of lipid peroxidation, causes mutations in bacterial and mammalian cells and cancer in rats. MDA reacts with deoxynucleosides in vitro and the monomeric adduct of MDA with deoxyguanosine (M1G-dR) is the major adduct formed. We have developed a sensitive analytical method to characterize and quantify M1G-dR from biological matrices using gas chromatography/electron capture negative chemical ionization mass spectrometry (GC/ECNCI MS). Reduction of M1G-dR with sodium borohydride produced a dihydro derivative (H2-M1G-dR). This more stable analog had improved high-performance liquid chromatographic characteristics which facilitated its isolation from biological fluids. H2-M1G-dR was converted to a monopentafluorobenzyl derivative with simultaneous depurination; it was then converted to the corresponding t-butyldimethylsilyl derivative and analyzed by GC/ECNCI MS. (2H2)H2-M1G was used as internal standard. Quantitative analysis was carried out using selected ion monitoring of m/z 302 and m/z 304 where the limit of detection was 10 pg (30 fmol) injected on-column. The level of M1G-dR in normal rat liver was 5.2 +/- 0.2 modified bases per 10(7) bases (n = 6 rats).

Ursodeoxycholic acid reduces the systemic toxicity of 1,2-dichloro,4-nitrobenzene by stimulating hepatic glutathione S-transferase in mice.

Male C57BL/6 mice were fed with normal diet (ND) or diets containing 0.3 or 0.5% ursodeoxycholic acid (UDCA) for 3 weeks. Glutathione S-transferase (GST) activities in the liver cytosolic fraction of these animals toward 1,2-dichloro,4-nitrobenzene (DCNB) as well as to 1-chloro,2,4-dinitrobenzene (CDNB) were significantly increased in a dose dependent manner in UDCA-treated groups compared with the control (ND-fed) animal group (one-way ANOVA). Reduced glutathione (GSH) levels tended to slightly decrease with UDCA diets but the difference did not attain a statistical significance (P > 0.05, one-way ANOVA). Twenty four hr survival rates after an oral challenge of 3.5 mg/kg of DCNB were significantly higher (P < 0.05, Chi-square test) in the two UDCA fed groups (10/10 for 0.5% group, 8/11 for 0.3% group) compared with the control group (3/11). Thus, UDCA appears to reduce the systemic toxicity of DCNB which is detoxified by the hepatic GST system. Although UDCA has been shown to exert hepatoprotective effects in experimental animals and humans in the past, to the best of our knowledge, the present study is the first report that UDCA reduces the systemic toxicity of a toxicant which is detoxified by the hepatic GST system. Although a direct proof is not available, it is most likely that the reduction of the systemic toxicity of DCNB was achieved by the increase in GST activity caused by UDCA feeding. This finding may open a new research field with regard to the unique biological properties of this bile salt in modulating hepatic detoxifying enzymes.

The optimal dosage of (-)deprenyl for increasing superoxide dismutase activities in several brain regions decreases with age in male Fischer 344 rats.

We previously reported that the optimal dosage of (-)deprenyl to increase superoxide dismutase (SOD) activities in striatum in rats differs 10 fold between young male and female rats (1). Furthermore, in female rats the optimal dosage increased with age (1). In the present study in order to clarify how the optimal dosage of this effect changes with age in male rats, we examined the effects of four different dosages of deprenyl on SOD enzyme activities in striatum and several other tissues in old (28-29-month-old) male Fischer 344 (F-344) rats. Continuous s.c. infusion of deprenyl for 3 wks increased activities of SOD and catalase (CAT) in striatum, substantia nigra and cortical regions but not in hippocampus, cerebellum or the liver. The dose of 0.5 mg/kg/day was found to be most effective, while higher (1.0, 2.0 mg/kg/day) or lower (0.1 mg/kg/day) dosages were less effective. This value of 0.5 mg/kg/day was 4 fold lower than the dosage of 2.0 mg/kg/day which was most effective in increasing SOD and CAT activities in young (5-7 month old) male rats of the same strain (1,2). The decline of the optimal dosage with age found in male rats is best explained by a possible decline with age in the hepatic microsomal monooxygenase enzyme activities that are involved with the metabolism of deprenyl. In view of the large differences in the optimal dosages shown among different sexes and ages of rats, future studies regarding the unique effect of this drug in prolonging the life span of rats must be carefully investigated with the caution in mind that the optimal dosage for the life prolonging effect may well differ depending on sex, age and possibly strain and species of animal model used.

Difference in response of hepatic glutathione S-transferase activities to protein-free diet between young and old C57/BL male mice.

Responses of hepatic glutathione S-transferase (GST) activities to protein-free diet (PFD) and normal diet (ND) refeeding were compared for young (6-month-old) and old (22-month-old) C57/BL male mice. Enzyme activities toward 1-chloro-2,4-dinitrobenzene (CDNB) were not significantly different between young and old rat livers in the basal condition without diet manipulation. When animals were fed PFD for 1 week, GST activities toward CDNB significantly declined in both age groups in comparison to respective basal values, but there was no significant difference in activities between the two age groups after a 7-day PFD. When they were refed with ND for 2 days (on day 2 of ND), the activities in young mice rose to a level significantly higher than the corresponding basal value. In contrast, in old animal livers, the activity slightly but further tended to decline on day 2 of ND. Activities in old rat livers returned to the basal level on day 5 of ND, while activities in young animal livers that increased to levels higher than basal levels due to the overshoot returned to the basal level on day 7 of ND. Enzyme activities toward 1,2-dichloro-4-nitrobenzene (DCNB) were significantly higher in young rat livers than in old ones at the basal period. However, enzyme activities also overshot the basal level on day 2 of ND after 7-day PFD in young mouse livers, while in old mouse livers the activities were lowest on this day. Activities returned to the basal level on day 7 of ND in both age groups. Thus, the greatest difference in enzyme activities between young and old mouse livers for both substrates was observed on day 2 of ND after 7-day PFD, rather than at either the basal period or immediately after 7-day PFD. The results essentially agree with our previous findings on female C57/BL mice as well as female Fischer-344 rats, suggesting that the age-induced changes in the GST system become clearly manifest after diet manipulation of PFD followed by ND refeeding, rather than in values during a basal period without diet manipulation, regardless of sex or species of animal.

(-)Deprenyl increases activities of superoxide dismutase and catalase in striatum but not in hippocampus: the sex and age-related differences in the optimal dose in the rat.

The dose of (-)deprenyl (2.0 mg/kg/day, sc, for 3 weeks) which significantly increased activities of superoxide dismutase (SOD) and catalase in the striatum of young male rats significantly reduced these activities in young female rats but did not change the SOD activity in old female rats. In order to clarify these effects, different doses of the drug were continuously infused sc for 3 weeks in three groups of rats (young males and young and old females). When a 10-fold smaller dose (0.2 mg/kg/day) was applied in young female rats, activities of both SOD and catalase were significantly increased, while a higher dose of 0.5 mg/kg/day was ineffective and a lower dose of 0.1 mg/kg/day was substantially less effective. In old female rats, doses of both 0.5 and 1.0 mg/kg/day were equally effective in elevating activities of SOD and catalase, while a lower dose of 0.1 mg/kg/day was less effective. The activity of glutathione peroxidase (GSH Px) remained unchanged in all groups, except for a significant decrease in the activity of non-selenium-dependent GSH Px in both young and old female rats given the highest drug dose (2.0 mg/kg/day). Furthermore, activities of all three enzymes remained unchanged in the hippocampus in most groups. The results indicate that (-)deprenyl significantly increases activities of both SOD and catalase in the striatum, but not in hippocampus of rats, and that the optimal dose is very different depending on the sex and age of the animal.

Differences in tolerance to hypoxia/anoxia in mice of different ages.

Tolerance to hypoxic stress was examined in male C57BL mice of different ages through use of a hypoxic chamber (4% oxygen, 96% nitrogen). The basal survival times in a hypoxic chamber did not significantly differ between mice of different ages (6 wks, 8 wks, 10 months, 28 months). The pretreatment of antihypoxic prostaglandin I2 (PGI2) significantly prolonged the survival time in both adult (10-month-old and aged (30-month-old) mice, but the effect tended to be smaller in aged mice. The effect of sublethal hypoxia treatment on survival time in a hypoxic chamber was minimal in both adult and aged mice, but the effect tended to be smaller in aged mice. IP injection of sublethal doses of potassium cyanide (3.0 mg/kg) significantly prolonged the survival time in a hypoxic chamber when examined 10 min after the pretreatment in immature mice (6 wks), but in adult and aged mice, the survival time tended to be slightly shorter at that time than respective control values without pretreatment. The maximal survival time after KCN pretreatment was longest in immature mice and shortest in aged mice. The results suggest that the basal tolerance to hypoxic stress in mice is little affected by aging. However, the potential antihypoxic mechanisms appear to be progressively affected by aging.

Age-related alterations of enzyme activities and subunits of hepatic glutathione S-transferases in male and female Fischer-344 rats.

Enzyme activities of glutathione S-transferases (GSTs) toward five different substrates (benzalacetone (PBO), styrene oxide (STOX), sulfobromophthalein (BSP), 1,2-dichloro-4-nitrobenzene (DCNB) and 1-chloro-2,4-dinitrobenzene (CDNB)) as well as concentrations of four subunits of GST isozymes (1, 2, 3 and 4) were determined using cytosol fractions obtained from livers of young (6 months) and old (26 months) Fischer-344 rats of both sexes. Values for enzyme activities for three substrates (DCNB, BSP and PBO) in young male rats were significantly higher than the corresponding values in female rats. In old male rats, values were generally lower than the corresponding values in young male rats, becoming close to corresponding values in young female rats. Old female rats, however, exhibited values close to those in young female rats, except for DCNB and STOX values, which were slightly lower in old female rats. GST subunits 3 and 4, as determined by high-performance liquid chromatography after purification by affinity chromatography using S-hexyl-glutathione, were predominant in young males, whereas concentrations of subunits 1 and 2 were higher in females than in males. In male rat livers, concentrations of subunits 3 and 4 decreased considerably with age while those of subunits 1 and 2 increased, so that the subunit pattern in old male rats tended to be similar to that of young female rats. In old females, a decrease in the concentration of subunits 3 and 4 and an increase in the concentration of subunit 1 were also observed as in old male rats, while the subunit 2 concentration tended to decline. Furthermore, the elution pattern of affinity chromatography changed with age, yielding an earlier elution of most subunits in old male rats and of subunit 1 in old female rats. The results suggest that age-related changes that occur with GSTs in livers of male rats are essentially a feminization of the isozyme pattern. However, despite rather unremarkable changes in enzyme activities with age in females, considerable changes of subunit pattern (a general decrease in concentration of subunits 2, 3 and 4 and an increase in the concentration of subunit 1) were also observed in female rats, and these were much greater than could be predicted from enzyme activity changes with age in this sex.

(-) deprenyl induces activities of both superoxide dismutase and catalase but not of glutathione peroxidase in the striatum of young male rats.

Daily s.c. injection of (-)deprenyl (2.0 mg/kg/day) for three weeks in young male rats caused a threefold increase in superoxide dismutase (SOD) activity in the striatum of the brain compared with the value in saline-injected control rats. Furthermore, the activity of catalase (but not of glutathione peroxidase) was also increased significantly by deprenyl treatment. The results confirmed the previous findings of Knoll on SOD activity and furthermore provided evidence that the activity of catalase is also significantly induced by the drug, which was not found in the previous study.

Effect of protein-free diet on activities and subunits of glutathione S-transferase in livers of young and aged female rats.

Female Fischer-344 rats of different ages (8 and 25 months old) were fed a protein-free diet (PFD) for 7 days and refed a normal diet (ND) (23% protein) thereafter. Rats were killed immediately after the PFD was stopped (day 0) and at different time intervals during refeeding of a ND. Four subunits (1,2,3 and 4) and activities of glutathione S-transferases (GSTs) toward five different substrates, [styrene oxide (STOX), 1,2-dichloro-4-nitrobenzene (DCNB), 1-chloro-2,4-dinitro benzene (CDNB), sulfobromophthalein (BSP) and benzalacetone (PBO)] were determined. There were no significant differences between young and old rats in the liver enzyme activities before the PFD. The PFD caused significant decreases in activities for three substrates (DCNB, BSP and STOX) in both age groups, with no significant differences between young and old rats a day 0. During recovery from the PFD, activities for the three substrates exceeded basal levels in young rats but at different time intervals (STOX, day 2; BSP, day 5; DCNB, day 9), while enzyme values in old rats tended to return slowly to basal values with no "overshoot." Concentrations of subunits 3 and 4 in young rat livers that were depressed by the PFD did not recover until day 9 of the ND, while subunits 2 and especially 1 increased during the ND refeeding, overshooting the basal levels. In contrast, in old rat livers the only change was a reduction of subunit 1 by the PFD and its gradual recovery during ND refeeding. These results demonstrate that our previous observation of overshooting of enzyme activities in mice is reproducible in rats but with certain substrate specificities. Furthermore, changes in subunit concentrations caused by aging and a PFD are more complex than what was predicted from changes in enzyme activities of GSTs.

Hepatic biotransformation and choleretic effect of 7-ketolithocholic acid in the rat.

Hepatic biotransformation and the effect on bile flow of 7-ketolithocholic acid (7-oxo-3 alpha-hydroxy-5 beta-cholan-24-oic acid), in comparison to ursodeoxycholic acid, were examined in rats under conditions of continuous infusion of solutions of sodium salts of these bile acids (1.2 mumol/min/100 g body wt) for 2 hr. Both bile salts elevated the bile flow rate as well as the bile bicarbonate concentration to a similar degree. The minor difference observed was a transient (10-20 min) and subtle drop of bile flow during the first hour in rats given 7-ketolithocholate. In ursodeoxycholate infused rats, the major bile salt in the bile was its taurine conjugate, although excretion of tauroursodeoxycholate dropped considerably during the second hour. In 7-ketolithocholate infused rats, the major bile salt in the bile was again its taurine conjugate, but ursodeoxycholate and chenodeoxycholate and their conjugates were also excreted. In contrast to ursodeoxycholate infused rats, the drop in excretion of taurine conjugates and the increase of glycine conjugates in rats infused with 7-ketolithocholate were more rapid. In rats infused with 7-ketolithocholate, excretion of ursodeoxycholate and its conjugates was significantly higher than the corresponding values for chenodeoxycholate, suggesting that 7-ketolithocholate is reduced predominantly to the 7 beta-epimer in this species. However, the concentration of ursodeoxycholate and its conjugates excreted into the bile in rats infused with 7-ketolithocholate was only 10% of that of rats infused with ursodeoxycholate, yet the magnitude of choleresis and the rise in bile bicarbonate concentration were similar in both experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-related differences in the coordination disturbance and anticonvulsant effect of oxazepam in mice.

The effects of oxazepam on coordination and maximal seizure were compared between young (6-month-old) and old (24-month-old) BDF1 mice by using a rotorod test and a pentylenetetrazole (PTZ)-induced seizure test, respectively. The apparent sensitivity to oxazepam's anticonvulsant effect, as examined by its effect on PTZ-induced maximal seizure, was increased in old animals of both sexes in comparison to young ones. The effect of oxazepam on coordination was also significantly greater in old male mice as compared with the young. The results of the present study, together with our past observations on other anticonvulsants such as phenytoin and phenobarbital, strongly suggest that the anticonvulsant effect, as well as the effect on coordination, in mice generally increases with age.

Identification of protein-bound riboflavin in rat hepatocyte plasma membrane as a source of autofluorescence.

The presence of flavin compound(s) giving a yellowish-green autofluorescence in rat hepatocyte plasma membrane has recently been reported (Nokubo, M. et al. (1988) Biochim. Biophys. Acta 939, 441-448). The fluorophore can quantitatively be extracted with water at 80 degrees C from isolated plasma membranes. Gel filtration of the extract eluted with water showed two peaks, the fluorescence of which closely resembled that of riboflavin. The major peak comigrated with proteins and the minor one displayed a position identical to authentic riboflavin. When the components of the major peak were rechromatographed after acetic acid treatment and eluted with 20 mM of acetic acid, the fluorescent compound separated from the proteins and eluted at the same position as riboflavin. In paper chromatography and HPLC, the behavior of the fluorescent compound (separated by acid treatment from the proteins) was identical to that of riboflavin. SDS gel filtration of subcellular fractions of rat liver revealed that riboflavin was the dominant flavin, whereas FAD and FMN were not detectable in the plasma membrane. Microsomes and mitochondria contain predominantly FAD and FMN, and only minor quantities of riboflavin. The presence of riboflavin in the plasma membrane is a novel finding, the functional significance of which is still unclear; however, a hypothesis can be forwarded on the basis of the ability of flavins to generate superoxide anion radicals during their autoxidation.

Morphological, physiological and biochemical alterations in livers of rodents induced by protease inhibitors: a comparison with old livers.

A "Protease inhibitor model of aging" has been proposed primarily based on observations on brain tissues exposed to a thiol protease inhibitor, leupeptin (Ivy et al., 1984a). In order to validate this model in terms of a mechanism of cellular aging, as well as of lipofuscin formation in particular, attempts have been made to induce lipofuscin in hepatocytes in young rodent (rat and mouse) livers by continuous i.p. infusion of two different thiol protease inhibitors, leupeptin and E-64C. With doses of leupeptin higher than 1.0 mg/100g/day for 2 wks, a fine granular lipofuscin-like deposition with distinct yellowish-green fluorescence was induced in young rat hepatocytes. The deposition became greater in degree with increasing leupeptin doses. In Kupffer cells and other endothelial cells, fluorescent granules were also induced. In contrast to rat livers, lipofuscin-like pigments induced in hepatocytes in mice were much less, even with a higher dose (20 mg/100 g/day). E-64C also induced the accumulation of lipofuscin-like pigments at a dose of 5 mg/100 g/day, their characteristics being very similar to those induced by leupeptin, but the accumulation being smaller in degree. The fluorescence of leupeptin induced lipopigments was yellowish-green having a peak around 520 nm in emission profile, closely resembling that observed in old rat livers. The hepatobiliary transport functions such as biliary transport maximum (Tm) for sulfobromophthalain and the biliary recovery of iv injected ouabain which are known to decline with age tended to decline in young (6-wk-old) rats administered with leupeptin at a dose of 5 mg/100 g/day for 2 wks. On the other hand, dolichol concentration in leupeptin treated livers was not increased in comparison to control livers, whereas in old rat livers, the dolichol concentration was more than 2 times greater than in young livers. A clear-dose-dependent deposition of ceroid-lipofuscin induced in young rodent livers by protease inhibitors strongly suggests that the "Protease inhibitor model" is generally valid not only for the brain but for other tissues such as the liver, and for two different thiol protease inhibitors.

Differences in the influence of diet on hepatic glutathione S-transferase activity and glutathione content between young and old C57 black female mice.

The effect of a protein-free diet (PFD) on hepatic activity of glutathione S-transferase (GST) and hepatic content of total glutathione (GSH) was examined in young (9-month-old), middle-aged (17-month-old) and old (27-month-old) C57BL/6CrS1c female mice. There were no significant differences in the control values of GSH or of enzyme activity for four of five substrates among young, middle-aged, and old animals fed normal diet (ND) only. Both GSH and GST activity were significantly decreased by the 7-day PFD in both young and old groups but the decrement was generally greater in old mice. After a 2-3-day refeeding of ND, young mouse enzyme activities were significantly higher than control (basal) values for all five substrates, whereas old mouse values were still significantly lower than corresponding control values. There was no overshooting of GSH levels after refeeding of ND in either young or old animals. This study indicates that an age difference in this detoxification system can be clearly demonstrated in the hepatic response to PFD feeding and especially to ND refeeding, despite the enzymes' stable basal activities with aging.

The neurotoxicity of phenobarbital and its effect in preventing pentylenetetrazole-induced maximal seizure in aging mice.

The effects of age on the neurotoxicity of phenobarbital and its anticonvulsant effect were examined in female BDF1 mice of different ages by means of a rotorod test and the pentylenetetrazole (PTZ)-induced maximal seizure, respectively. The minimal neurotoxic concentrations (MTCs) of phenobarbital in both plasma and brain evaluated by a rotorod test were 50% lower (p less than 0.05) in old (24-months-old) mice compared with the respective values in young (6-months-old) mice, while 12-months values were the highest of the three groups. Mice given some dosages of phenobarbital, particularly old (24-months-old) mice, died within the 15-min observation period after an i.p. injection of PTZ (150 mg/kg) without demonstrating a hindlimb extensor component (HLE) of maximal seizure. When these animals were classified as responders with regard to the anticonvulsant effect of phenobarbital, the minimal effective concentrations (MECs) in plasma and brain required for abolishing the PTZ-induced maximal seizure in old mice were only 10-20% those of young mice. Present results coupled with our past studies using electroshock seizure suggest that both the neurotoxicity and the efficacy of phenobarbital increase with age in mice.

Characterization of the autofluorescence of rat liver plasma membranes.

The autofluorescence of isolated rat liver cell plasma membranes was characterized in vitro in relation to the autofluorescence used previously for fluorescence recovery after photobleaching (FRAP) studies. The fluorescence of membrane preparations displayed an emission pattern with a maximum at around 525 nm when excited with a 468 nm blue light. The excitation spectrum monitored at 525 nm closely resembled that of flavin compounds (riboflavin, FAD, FMN). The chloroform extract of the membrane fraction showed practically no fluorescence, whereas, both the water-soluble and water-insoluble protein fractions remaining after chloroform extraction were strongly fluorescent. The fluorescence disappeared almost completely under the effect of sodium hydrosulfite, and recovered after oxidation either by shaking in air or by adding buffered hydrogen peroxide solution. The fluorescence of the acid extract of the plasma membranes photolyzed in an alkaline medium was quite similar to that of lumiflavin obtained from the photolysis of riboflavin in an alkaline medium. The plasma membranes prepared from isolated hepatocytes (which were completely devoid of endothelial cell contamination) exhibited the same autofluorescence in the liver cell plasma membranes. The results suggest that the autofluorescence of the liver cell plasma membranes is most likely of a character similar to that of flavin, bound to hepatocyte plasma membrane proteins. This fluorescence is suitable for measuring the average lateral diffusion constant of proteins by means of FRAP methods.

The age-dependent decline in the biliary transport maximum of conjugated sulfobromophthalein in the rat.

In order to elucidate the effect of aging on the biliary transport of organic anions, we examined the biliary transport maximum (Tm) for sulfobromophthalein (BSP) conjugated with glutathione in male and female Fischer-344 rats of various ages (3, 6, 24 and 30 months) during the constant intravenous infusion of conjugated BSP. For young rats the Tm values for conjugated BSP were higher in females than in males in contrast to the reported higher Tm value in males infused with free (unconjugated) BSP. In both sexes, Tm values progressively decreased with age up to 30 months. The results suggest that the previously reported higher Tm value for young male rats compared to young females during free BSP infusion is due to the lower conjugation capacity in females, and that the biliary transport process per se is more efficient in females at least for organic anions like conjugated BSP. Furthermore, the results support the previously proposed hypothesis that the biliary transport mechanism per se is steadily affected by aging in rats of both sexes.