Increase in non-transferrin bound iron and the oxidative stress status in epilepsy patients treated using valproic acid monotherapy.

OBJECTIVE: This study aims to investigate the alteration of iron homeostasis and oxidative stress status in epilepsy patients treated with valproic acid (VPA) monotherapy. MATERIALS: 24 epilepsy patients receiving VPA monotherapy (12 men, 12 women, age 27.5 ± 7.2 y) and 24 sex- and age-matched healthy volunteers were included in the study. METHODS: The level of iron status parameters; serum iron, ferritin, transferrin saturation, non-transferrin bound iron (NTBI), serum level of trace elements (copper, zinc and selenium), concentration of antioxidant parameters, activities of antioxidant enzymes and level of lipid peroxidation product were determined. RESULTS: NTBI was found in the patients although their other iron status parameters were normal. Levels of antioxidant parameters were decreased while activities of antioxidant enzymes were increased. Levels of serum MDA were significantly increased in patients with epilepsy. The daily dose of valproic acid associated was statistically significant: serum concentration of NTBI (r = 0.579; p = 0.003) and MDA (r = 0.465; p = 0.022). A positive correlation existed between NTBI and zinc (r = 0.522; p = 0.009). CONCLUSION: According to our results, VPA treatment in patients with epilepsy contributes to the metabolism of iron, leading to the formation of NTBI and an increase in oxidative stress.

Urinary 6-hydroxymelatonin sulfate excretion in intellectually disabled subjects with sleep disorders and multiple medications: validation of measurements in urine extracted from diapers.

The aim of this study was to examine the applicability of urinary 6-hydroxymelatonin sulfate (MT6s) measurements in the evaluation of melatonin secretion in intellectually disabled patients with sleep disorders. All 17 patients received drugs with potential interactions with melatonin metabolism. Serum melatonin 24-h profiles were determined at hourly intervals. The area under the curve (AUC) value, peak amplitude, half-rise time, and half-decline time were calculated individually. Urinary MT6s excretion was determined from samples collected from disposable diapers during three consecutive days at varying intervals. The average excretion rate for each hour of the day was calculated. The excretion profiles were characterized by total amount of MT6s excretion/24 h/kg body mass, amount of excreted MT6s during 6 h of maximum excretion (MAX 6h), and start time of the maximum excretion (start MAX 6h). There were significant positive correlations between serum melatonin AUC value and total excretion of MT6s/body mass, between serum melatonin amplitude and urinary MAX 6h, and between melatonin half-rise time and start MAX 6h; one patient on phenobarbital medication was out of line. The serum melatonin profiles of the patients were classified by comparing them with those of matched healthy volunteers (low-, normal-, or high secretors, normal or delayed rhythm). Similarly, the parameters of MT6s profiles were compared with those obtained from healthy controls, and the patients were reclassified as normal or aberrant. The classifications based on serum melatonin and urinary MT6s measurements were mostly concordant. The daily pattern of urinary MT6s excretion reliably reflected the phase of the serum melatonin rhythm irrespective of the medications, but in some cases, the total amount of excreted MT6s was lower than expected based on serum melatonin measurements.

Evidence for the presence of cytochrome P-450 in mastitic bovine mammary gland.

The content of cytochrome P-450 in mastitic and healthy lactating bovine mammary gland was analyzed. Evidence for the existence of cytochrome P-450 in mastitic tissues was found. The amount of cytochrome P-450 formed in the assay ranged from 0.017 to 0.031 nmole/mg protein. The highest concentrations were found in severely inflamed samples, whereas and traces or no cytochrome P-450 were found in healthy tissues. The significance of cytochrome P-450 in mastitic mammary gland is discussed in relation to host defence involving oxidative killing of pathogens.

Coumarin 7-hydroxylase in inbred strains of mice: comparison with other microsomal monooxygenase activities and induction by pyrazole.

This study was carried out in order to find out the effects of pyrazole on liver drug metabolism in several inbred mice: D2, B6, BALB, and AKR and in the outbred mouse NMRI. Compared to control pyrazole treatment decreased the microsomal cytochrome P-450 content of liver to 60-70% and benzo(a)pyrene hydroxylase and ethylmorphine N-demethylase activities to 40-55% and increased 7-ethoxycoumarin O-deethylase activity to 100-200% in AKR, BALB, B6, and NMRI mice and 300% in D2 mouse. Coumarin 7-hydroxylase was increased only 200-300% in B6, AKR, and BALB mice and as much as 700% in D2 and 900% in NMRI mice compared to control. Coumarin 7-hydroxylase is under different genetic control from other monooxygenase activities and differently expressed in various strains of mice.

Inhibition by methylglyoxal bis(guanylhydrazone) of drug oxidation reactions catalyzed by mouse liver microsomes in vivo and in vitro.

The activity of coumarin 7-hydroxylase (coumarin 7-hydroxylation) was inhibited in B6 mouse liver after a single injection of methylglyoxal bis(guanylhydrazone (MGBG). The decrease in the activity in vivo was greatest (70%) one day after the drug injection and the hydroxylase activity in microsomal fraction prepared from livers of MGBG-treated B6 mice was still 25% decreased 5 days after the drug. The amount of cytochrome P-450 also was decreased in MGBG-treated livers with the same time-dependency as the inhibition of coumarin 7-hydroxylation. MGBG and its close derivative 1,1'-[methylethanediylidene)dinitrilo)bis(3-aminoguanidine) (MBAG) inhibited the activity in vitro of coumarin 7-hydroxylase, benzo(a)pyrene hydroxylase and 7-ethoxy 0-de-ethylase when microsomes were prepared from livers of untreated B6 mice. In every case MBAG was a better inhibitor than MGBG in vitro. The in vitro inhibition of MGBG of several drug metabolizing enzymes was not reversed when microsomes were preincubated with 1 mM putrescine, spermidine or spermine. These results suggest that the anti-cancer drug, MGBG, has a severe effect(s) on the drug metabolizing system at concentrations reached during the treatment of patients with MGBG.

Selective induction of coumarin 7-hydroxylase by pyrazole in D2 mice.

Pyrazole, was given to DBA/2N (D2), C57BL/6N (B6) and AKR/N mice to study its effects on hepatic drug metabolism. A decrease in the total amount of microsomal cytochrome P-450 as well as in the activities of ethylmorphine demethylase and benzo[a]pyrene hydroxylase was found. On the other hand ethoxycoumarin de-ethylase was increased 1.5-2.5-fold (depending on the strain of mouse) and coumarin 7-hydroxylase as much as sevenfold (but only in D2 mice) after pyrazole treatment. This increase was much higher than that caused by phenobarbital, the only well known inducer of coumarin 7-hydroxylase. By reconstituting the mono-oxygenase complex after purification of cytochrome P-450 we found a 40-fold increase in coumarin 7-hydroxylase and eightfold increase in ethoxycoumarin de-ethylase after pyrazole treatment. This was found only in D2 mice. An antibody previously developed against a cytochrome P-450 fraction from the the D2 strain with a high coumarin 7-hydroxylase activity inhibited the microsomal coumarin 7-hydroxylase almost 100% after pyrazole pretreatment of the animals. In the case of control or phenobarbital-treated mice the inhibition was somewhat weaker. With the reconstituted mono-oxygenase complex the inhibition of coumarin 7-hydroxylase was almost 100% both for control and pyrazole-treated D2 mice. The data indicate that pyrazole causes an induction of the microsomal monooxygenase complex different from that caused by phenobarbital or 3-methylcholanthrene and selective for coumarin 7-hydroxylation or 7-ethoxycoumarin de-ethylation. This induction was strong in D2, weak in B6 and absent in AKR/N mice.

Comparison of kidney, lung and liver coumarin 7-hydroxylases in phenobarbital pretreated DBA/2J and C57BL/6J mice.

In this paper we describe catalytic and immunological properties of coumarin 7-hydroxylase, a cytochrome P-450 dependent enzyme activity, in the liver, kidney and lung of C57BL/67 and DBA/2J mice. Coumarin 7-hydroxylase activity was higher in D2 than in B6 mice in all three organs. For both strains of mice, liver had the highest enzyme activity when expressed per milligram of microsomal protein. However, when expressed per nmole cytochrome P-450 there was no difference in the enzyme activity between the tissues. Inhibition of microsomal coumarin 7-hydroxylase by antibodies previously developed in our laboratory against a cytochrome P-450 fraction from D2 and B6 mouse liver, associated with coumarin 7-hydroxylase, occurred as follows. In D2 mice both antibodies caused approximately 50% inhibition of the enzyme activity of all three organs. In B6 mice, however, the only organ where considerable inhibition took place was the liver, and only when antibody against B6 cytochrome P-450 was used. Ouchterlony immunodiffusion analysis revealed a 100% crossreactivity between the two strains of mice when similar organs were compared. The 100% crossreactivity was also found between the liver and lung in both strains of mice. However, only a 50% crossreactivity was found between kidney and liver or kidney and lung in B6 and between the kidney and lung in D2. The data demonstrate interorgan and interstrain differences in the immunological and catalytical properties of cytochrome(s) P-450 catalysing coumarin 7-hydroxylation.

Catalytic and immunological comparison of coumarin 7-hydroxylation in different species.

Coumarin 7-hydroxylation activities were determined in the liver microsomes of eight different species: pig, mouse (three strains), rat, hamster, guinea-pig, rabbit, rainbow trout and crayfish. A high coumarin 7-hydroxylase activity was found in the microsomes of D2 mouse, rabbit, hamster and pig, an intermediate activity in case of B6 and AKR mice, rat and guinea-pig and a very low activity in rainbow trout and crayfish. In Ouchterlony immunodiffusion analysis our antibody against coumarin 7-hydroxylase specific cytochrome P-450 from D2 mice was able to form a weak precipitin line only with rat and hamster microsomes in addition to the three strains of mice (D2, B6, AKR) where the band was very sharp. Strongest inhibition by the antibody (50% or more) of microsomal coumarin 7-hydroxylase was found in the case of the three strains of mice and rabbit. In the case of the other species the inhibition was very weak or did not exist.

Purification and characterization of a microsomal cytochrome P-450 with high activity of coumarin 7-hydroxylase from mouse liver.

Phenobarbital-induced coumarin 7-hydroxylase is high in DBA/2J and low in C57BL/6N inbred mice; this genetic difference is encoded by the Coh locus on chromosome 7. The aim of this study was to develop an antibody specific for this cytochrome P-450 polymorphism. P-450 fractions, highly specific for phenobarbital-inducible coumarin 7-hydroxylase activity, were purified from DBA/2J and C57BL/6N mouse liver microsomes. Both proteins are 49 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Soret peaks of the reduced cytochrome . CO complexes are 451 nm. Reconstituted DBA/2J coumarin 7-hydroxylase activity exhibits a V twice as high as, and a Km value 10-fold less than, the reconstituted C57BL/6N activity. Antibodies were raised in rabbit. By Ouchterlony immunodiffusion, both antibodies show 100% cross-reactivity with DBA/2J and C57BL/6N microsomes and purified antigens. Yet, DBA/2J but not C57BL/6N 7-hydroxylase activity is inhibited by the antibody to DBA/2J P-450. Both DBA/2J and C57BL/6N activities are blocked by the antibody to C57BL/6N P-450. Neither antibody has any effect on liver microsomal d-benzphetamine N-demethylase, ethylmorphine N-demethylase, aminopyrine N-demethylase, 7-ethoxycoumarin O-deethylase, acetanilide 4-hydroxylase, or aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity. The DBA/2J protein most specific for phenobarbital-induced coumarin 7-hydroxylation is designated 'P-450Coh'. Anti-(P-450Coh) precipitates a relatively minor 49-kDa protein from detergent-solubilized microsomes and from in vitro translation of poly(A+)-enriched total RNA of phenobarbital-treated DBA/2J mouse liver, whereas the major phenobarbital-induced P-450 proteins exhibit a molecular mass of about 51 kDa. The immunoprecipitated translation products correspond to a messenger RNA of 2100 +/- 100 nucleotides.