Decrease in tetrahydrobiopterin as a possible cause of nephropathy in type II diabetic rats.

A decrease in renal synthesis of nitric oxide (NO) in the progression of diabetic nephropathy has been documented. As (6R)-5,6,7,8-tetrahydrobiopterin (BH4) is an essential cofactor of NO synthase, we investigated whether BH4 deficiency is involved in the pathogenesis of nephropathy. Ten-week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats were used as a type II diabetic model, and Long-Evans Tokushima Otsuka (LETO) rats as the healthy controls. OLETF rats were orally treated with BH4 (10 mg/kg daily) or with water from 10 to 61 weeks of age. In another experiment, OLETF rats were treated orally with a calcium channel blocker, benidipine (5 mg/kg daily), or with 0.3% carboxymethyl cellulose (nontreated) from 10 to 52 weeks of age. Proteinuria was observed periodically, and at the end of the study, BH4 level and GTP cyclohydrolase I (GTPCH) activity in the kidney were measured. Proteinuria was observed at 13 weeks of age in the OLETF rats, and deteriorated until 61 weeks of age. Supplemental BH4 reduced the proteinuria. At 52 weeks of age, GTPCH activity and the BH4 level were decreased in the plasma and kidneys of OLETF rats, whereas they were significantly higher in the benidipine group than in the nontreated group. Proteinuria was milder in the benidipine group than in the nontreated group, without a concomitant decrease in blood pressure. Histologically observed glomerulosclerosis was mild in the BH4 and benidipine groups. In type II diabetic rats, renal BH4 is considered to play a crucial role in the pathogenesis of diabetic nephropathy. Benidipine was found to preserve BH4 levels, suggesting therapeutic renoprotective effects.

Interindividual variation in the ratio between plasma morphine and its metabolites in cancer patients.

In 25 cancer patients treated with slow-release oral morphine and in 10 cancer patients treated with continuous infusion of morphine, plasma steady-state concentrations of morphine (M), morphine-3-glucuronide (M-3-G) and morphine-6-glucuronide (M-6-G) were determined by high-performance liquid chromatography. Blood samples were withdrawn at 0, 2 and 6 h after oral administration in patients treated with slow-release oral morphine and once or twice a day in patients treated with continuous infusion of morphine. In four cancer patients treated with continuous infusion of morphine, in order to analyze chronopharmacokinetic variability, the M-3-G/M ratio was observed at 12:00 h and 24:00 h. No significant changes were observed in M-3-G/M ratios and M-6-G ratios at 0, 2, and 6 h after oral administration of morphine. The M-3-G/M ratio (38.6 +/- 25.7) in the oral morphine group was significantly higher than that (15.3 +/- 12.9) in the continuous infusion group (p < 0.01). There was an approximately 10-fold interindividual variation in the M-3-G/M ratio both in the continuous infusion group and in the oral morphine group. These results suggest that the activity of UDP glucuronosyltransferase 2B7 in the intestinal metabolism of morphine may play an active part in a large interindividual variation in the ratio of metabolites to morphine. Further studies are needed to clarify this hypothesis.

Coronary endothelial dysfunction in the insulin-resistant state is linked to abnormal pteridine metabolism and vascular oxidative stress.

OBJECTIVES: We investigated whether abnormal pteridine metabolism is related to coronary endothelial dysfunction in insulin-resistant subjects. BACKGROUND: Depletion of tetrahydrobiopterin (BH(4)) and elevation of the 7,8-dihydrobiopterin (BH(2)) (activating and inactivating cofactors of nitric oxide synthase [NOS], respectively) contribute to impairment of NO-dependent vasodilation through reduction of NOS activity as well as increased superoxide anion generation in insulin-resistant rats. METHODS: Thirty-six consecutive nondiabetic, normotensive and nonobese subjects with angiographically normal coronary vessels were studied. Traditional coronary risk factors, plasma pteridine levels, activities of erythrocyte dihydropteridine reductase (DHPR), the recycling enzyme that converts BH(2) to BH(4) and lipid peroxide (LPO) levels were measured and coronary endothelial function was assessed with graded infusions of acetylcholine (ACh). RESULTS: When we divided patients into tertiles based on insulin sensitivity, we observed stepwise decreases in the maximal ACh-induced vasodilation and plasma BH(4)/7,8-BH(2) ratio, and increases in coronary LPO production as insulin sensitivity decreased. The ACh-induced vasodilation was positively correlated with insulin sensitivity, BH(4)/7,8-BH(2) ratio and DHPR activity. Furthermore, BH(4)/7,8-BH(2) was inversely correlated with DHPR activity and insulin sensitivity. In multiple stepwise regression analysis, BH(4)/BH(2) was independently related to ACh-induced vasodilation and accounted for 39% of the variance. However, no significant correlation existed between other traditional risk factors and BH(4)/7,8-BH(2). CONCLUSIONS: These results indicate that both abnormal pteridine metabolism and vascular oxidative stress are linked to coronary endothelial dysfunction in the insulin-resistant subjects.

Different effects of St John's wort on the pharmacokinetics of simvastatin and pravastatin.

OBJECTIVE: St John's Wort, a widely used herbal product, is an inducer of CYP3A4 and it decreases blood concentrations of CYP3A4 substrates. The effects of St John's Wort on the pharmacokinetics of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors simvastatin (an inactive lactone pro-drug) and pravastatin were determined in this study. METHODS: Sixteen healthy male subjects (n = 8 in group 1 and n = 8 in group 2) took a St John's Wort caplet (300 mg) or matching placebo three times a day for 14 days in a double-blind, crossover study. On day 14, a single oral dose of 10 mg simvastatin and 20 mg pravastatin was given to subjects in group 1 and group 2, respectively. Blood samples were obtained during a 24-hour period after the administration of each drug. RESULTS: Repeated St John's Wort treatment tended to lower plasma simvastatin concentration and significantly (P <.05) lowered concentrations of simvastatin hydroxy acid, its active metabolite. The peak concentration in plasma (ratio, 0.72 of placebo) of simvastatin hydroxy acid tended to be decreased and its area under the plasma concentration-time curve between time zero and 24 hours after administration (ratio, 0.48 of placebo) was significantly decreased (P <.05) by St John's Wort. On the other hand, St John's Wort did not influence plasma pravastatin concentration. No significant differences were observed in the elimination half-life of simvastatin or pravastatin between the placebo and St John's Wort trials. CONCLUSIONS: This study showed that St John's Wort decreases plasma concentrations of simvastatin but not of pravastatin. Because simvastatin is extensively metabolized by CYP3A4 in the intestinal wall and liver, which are induced by St John's Wort, it is likely that this interaction is partly caused by the enhancement of the CYP3A4-mediated first-pass metabolism of simvastatin in the small intestine and liver.

Close correlation of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate, an intracellular active metabolite, to the therapeutic efficacy of N(4)-behenoyl-1-beta-D-arabinofuranosylcytosine therapy for acute myelogenous leukemia.

N(4)-Behenoyl-1-beta-D-arabinofuranosylcytosine (BHAC), a prodrug of 1-beta-D-arabinofuranosylcytosine, is used effectively for the treatment of leukemia in Japan. BHAC therapy may be more effective if it is delivered in conjunction with monitoring of 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP), the intracellular active metabolite of ara-C derived from BHAC. However, previous monitoring methods for ara-CTP were insufficiently sensitive. Here, using our new sensitive method, we evaluated the ara-CTP pharmacokinetics in relation to the therapeutic response in 11 acute myelogenous leukemia patients who received a 2-h infusion of BHAC (70 mg / m(2)) in combination remission induction therapy. ara-CTP could be monitored at levels under 1 mM. BHAC maintained effective levels of plasma ara-C and intracellular ara-CTP for a longer time, even compared with historical values of high-dose ara-C. The area under the concentration-time curve of ara-CTP was significantly greater in the patients with complete remission than in the patients without response. This greater amount of ara-CTP was attributed to the higher ara-CTP concentrations achieved in the responding patients. There was no apparent difference of plasma ara-C pharmacokinetics between the two groups. Thus, for the first time, the ara-CTP pharmacokinetics was evaluated in relation to the therapeutic effect of BHAC, and the importance of ara-CTP was proven. Administration of optimal BHAC therapy may require monitoring of the ara-CTP pharmacokinetics in each individual patient.

Monitoring of intracellular 1-beta-D-arabinofuranosylcytosine 5'-triphosphate in 1-beta-D-arabinofuranosylcytosine therapy at low and conventional doses.

1-beta-D-Arabinofuranosylcytosine (ara-C) is used empirically at a low, conventional, or high dose. Ara-C therapy may be optimal if it is directed by the clinical pharmacokinetics of the intracellular active metabolite of ara-C, 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (ara-CTP). However, ara-CTP has seldom been monitored during low- and conventional-dose ara-C therapies because detection methods were insufficiently sensitive. Here, with the use of our newly established method (Cancer Res., 56, 1800 -- 1804 (1996)), ara-CTP was monitored in leukemic cells from acute myelogenous leukemia patients receiving low- or conventional-dose ara-C [subcutaneous ara-C administration (10 mg / m(2) ) (3 patients), continuous ara-C infusion (20 or 70 mg / m(2) / 24 h) (7 patients), 2-h ara-C infusion (70 mg / m(2) ) (4 patients), and 2-h infusion of N(4)-behenoyl-1-beta-D-arabinofuranosylcytosine, a deaminase-resistant ara-C derivative (70 mg / m(2) ) (6 patients)]. Ara-CTP could be determined at levels under 1 microM. There was a close correlation between the elimination half-life values of the plasma ara-C and the intracellular ara-CTP. The presence of ara-C in the plasma was important to maintain ara-CTP. The continuous ara-C and the 2-h N(4)-behenoyl-1-beta-D-arabinofuranosylcytosine infusions maintained ara-CTP and the plasma ara-C longer than the subcutaneous ara-C or the 2-h ara-C infusion. They also afforded relatively higher ara-CTP concentrations, and consequently produced ara-CTP more efficiently than the 2-h ara-C infusion. Different administration methods produced different quantities of ara-CTP even at the same dose.

[The amount of the loss of cyclosporine A dose correlated with the amount of leaching di (2-ethylhexyl) phthalate from polyvinyl chloride infusion tube].

An interaction between cyclosporine A (CyA) injection and infusion tubes were examined. We used polyvinyl chloride (PVC) and polybutadiene (PB) tubes. CyA injection (Sandimmun) was diluted (0.495 mg CyA/ml) with saline and dripped through infusion tubes. The amounts of unsolved substances, loss of CyA dose and leached di (2-ethylhexyl) phthalate (DEHP) during the drip study were compared. CyA was not lost into the PB tube and no DEHP was leached. Therefore, using PVC tube, 11.9 mg of CyA were lost with in 24 h after the beginning of the administration, and the concentration of leached DEHP amounted to 93.6 micrograms/ml at 12 h. We also investigated the effects of the component of the einfusion solution on the loss of CyA into PVC tube using saline, electrolyte maintenance solution, 5% glucose and 10% maltose. Sugar-containing solutions were found to have less effects than other solutions on the loss of CyA dose and DEHP leaching. The leaching of DEHP may be a major factor for the generation of unsolved substances and the loss of CyA dose. In the clinical use of CyA injection, PB tube is the best selection and the sugar-containing solution is a second selection when PB infusion tubes are hard to obtain.

Alterations of tetrahydrobiopterin biosynthesis and pteridine levels in mouse tissues during growth and aging.

In the present study, we investigated age-related changes in pteridine levels and enzymatic activity responsible for tetrahydrobiopterin biosynthesis in mouse tissues. Until about 15 weeks after the birth, the remarkable change of tetrahydrobiopterin (BH4) was observed in all tissues tested. Between 20 and 50 weeks after the birth, pteridines levels were almost constant in all of the tissues. Total biopterin levels were decreased and levels of pterin and neopterin were increased in the period exceeding 50 weeks in all of the tissues. Activities of guanosine triphosphate (GTP) cyclohydrolase I, pyrvoyltetrahydropterin synthase, and the production of BH4 were recognized by specific biochemical assays, and we investigated the age-related changes in mouse tissues. The alteration of these enzymatic activities was indicated to be similar to that described in the change of pteridine levels.

Oral administration of tetrahydrobiopterin prevents endothelial dysfunction and vascular oxidative stress in the aortas of insulin-resistant rats.

We have reported that a deficiency of tetrahydrobiopterin (BH(4)), an active cofactor of endothelial NO synthase (eNOS), contributes to the endothelial dysfunction through reduced eNOS activity and increased superoxide anion (O(2)(-)) generation in the insulin-resistant state. To further confirm this hypothesis, we investigated the effects of dietary treatment with BH(4) on endothelium-dependent arterial relaxation and vascular oxidative stress in the aortas of insulin-resistant rats. Oral supplementation of BH(4) (10 mg. kg(-1). d(-1)) for 8 weeks significantly increased the BH(4) content in cardiovascular tissues of rats fed high levels of fructose (fructose-fed rats). Impairment of endothelium-dependent arterial relaxation in the aortic strips of the fructose-fed rats was reversed with BH(4) treatment. The BH(4) treatment was associated with a 2-fold increase in eNOS activity as well as a 70% reduction in endothelial O(2)(-) production compared with those in fructose-fed rats. The BH(4) treatment also partially improved the insulin sensitivity and blood pressure, as well as the serum triglyceride concentration, in the fructose-fed rats. Moreover, BH(4) treatment of the fructose-fed rats markedly reduced the lipid peroxide content of both aortic and cardiac tissues and inhibited the activation of 2 redox-sensitive transcription factors, nuclear factor-kappaB and activating protein-1, which were increased in fructose-fed rats. The BH(4) treatment of control rats did not have any significant effects on these parameters. These results indicate that BH(4) augmentation is essential for the restoration of eNOS function and the reduction of vascular oxidative stress in insulin-resistant rats.

Abnormal biopterin metabolism is a major cause of impaired endothelium-dependent relaxation through nitric oxide/O2- imbalance in insulin-resistant rat aorta.

To investigate underlying mechanisms responsible for the impaired nitric oxide (NO)-dependent vascular relaxation in the insulin-resistant state, we examined production of both NO and superoxide anion radical (O2-) and those modulating factors in aortas obtained from normal (CTR), insulin-treated (INS), or high fructose-fed (FR) rats. FR rats showed insulin resistance with endogenous hyperinsulinemia, whereas INS rats showed normal insulin sensitivity. Only FR aortic strips with endothelium elicited impaired relaxation in response to either acetylcholine or calcium ionophore A23187. Endothelial NO synthase (eNOS) activity and its mRNA levels were increased only in vessels from INS rats (P < 0.001), whereas eNOS activity in FR rats was decreased by 58% (P < 0.05) when compared with CTR rats. NO production from aortic strips stimulated with A23187 was significantly lower in FR than CTR rats. In contrast, A23187-stimulated O2- production was higher (P < 0.01) in FR than CTR rats. These differences were abolished when aortic strips were preincubated in the media including (6R)-5,6,7,8-tetrahydrobiopterin (BH4), an active cofactor for eNOS. Furthermore, as compared with CTR rats, aortic BH4 contents in FR rats were decreased (P < 0.001), whereas the levels of 7,8-dihydrobiopterin, the oxidized form of BH4, were increased, with opposite results in INS rats. These results indicate that insulin resistance rather than hyperinsulinemia itself may be a pathogenic factor for decreased vascular relaxation through impaired eNOS activity and increased oxidative breakdown of NO due to enhanced formation of O2- (NO/O2- imbalance), which are caused by relative deficiency of BH4 in vascular endothelial cells.

A modified fluorescence polarization immunoassay method incorporating fat emulsion (FE-FPIA) to determine cyclosporin A concentrations in rat skin.

We have developed a simple, sensitive and reliable assay procedure for cyclosporin A (CyA), a modified fluorescence polarization immunoassay method incorporating fat emulsion (FE-FPIA), to determine the CyA content in rat skin. The conventional fluorescence polarization immunoassay (FPIA) method for CyA using a commercially available FPIA kit, TDX cyclosporine monoclonal whole blood, was modified. A fat emulsion for intravenous infusion, Intralipos, was incorporated for dissolving the CyA extracted from the skin tissue, and a mixture of MeOH/purified water was used as the sample pretreatment medium instead of the precipitation reagent in the conventional FPIA kit intended for whole blood samples. These modifications enabled us to produce a reliable and the sensitive assay of CyA in skin tissue. The reproducibility (coefficient of variation), detection limit, and assay time for FE-FPIA were below 2%, 25 ng/ml, and about 24 min/24 samples, respectively, and were comparable with those for the whole blood samples determined by the conventional FPIA. Pre-purification of samples required by the HPLC assay is not needed in the FE-FPIA method. The usefulness of the FE-FPIA method in evaluating the topical pharmacokinetics of CyA in skin is discussed.

Complexes of serine acetyltransferase and isozymes of cysteine synthase in spinach leaves.

Polyclonal antibodies against cysteine synthase (CSase; EC 4.2.99.8) isozymes 1, 2, and 3 were used for the detection of complexes of these isozymes with serine acetyltransferase (SATase; EC 2.3.1.30). SATase was partially purified and found to complex with these isozymes by western blotting and immunotitration. When the complexes were treated with a high concentration of O-acetyl-L-serine, they did not dissociate. However, some complexes with CSase 1 or 3 dissociated when left for 24 h at 4 degrees C. Results of western blotting on SDS-PAGE showed that CSase 2 complexed with SATase. CSases 1, 2, and 3 all could complex with SATase, but the tightness of the bond differed.

Purification and characterization of a novel cysteine synthase isozyme from spinach hydrated seeds.

A novel type of cysteine synthase (CSase, EC 4.2.99.8) isozyme, designated as CSase 1', was purified to homogeneity from hydrated spinach seeds. The enzyme had a molecular weight of 68,000 and consisted of two identical subunits of M(r), 34,000. The apparent K(m) for O-acetyl-L-serine was 8.33 mM and that for sulfide was 0.66 mM. The activity of CSase 1' was maintained when it was treated at 60 degrees C for 1 min. This novel enzyme was similar to CSases 1, 2, and 3 already purified from spinach leaves, in results of double immunodiffusion, molecular weight, subunit composition, K(m) values for O-acetyl-L-serine and sulfide, and heat stability. On the other hand, N-terminal amino acid sequence, effects of immunotitration, pH optimum, and effects of hydroxylamine on purified CSase 1' were different from those of the other CSases. Furthermore, it was found that CSases 2S and 3S isolated from hydrated spinach seeds were identical with the CSases 2 and 3 reported previously. It was also disclosed that CSases 1, 2, and 3 were localized in chloroplasts, cytosol, and mitochondria, respectively.

[Bayesian forecasting of plasma vancomycin concentration using time-dependent pharmacokinetics].

A new prediction method of vancomycin (VCM) pharmacokinetics has been developed using the modified Bayesian forecasting method involved in time-dependent pharmacokinetics in clearance. We investigated to evaluate the usefulness of this new prediction method compared with that of the ordinary Bayesian forecasting method. Serum samples, obtained from 4 patients at least 3 different days during the period for the VCM treatment were assayed by fluorescence polarization immunoassay. VCM pharmacokinetic parameters and predicted serum VCM concentrations were calculated using this new method and the ordinary one according to the one-compartment model. The precision of the predicted serum VCM concentrations by these two methods at the third experimental day were evaluated with the mean prediction error (ME), mean absolute prediction error (MAE) and root mean squared error (RMSE). The most precise and least-bias prediction of serum VCM concentrations were observed using this new prediction method (ME: -0.36 +/- 1.40, MAE: 1.13 +/- 0.82 and RSME: 1.37). The time-dependent decrease of VCM clearance was observed in all patients. Therefore, the fitting of the actual serum VCM concentrations obtained using the ordinary method produced less precise results than that using this new method. These results suggest the usefulness of this new prediction method considering time-dependent changes in VCM clearance.

Comparative studies on cysteine synthase isozymes from spinach leaves.

Three types of cysteine synthase (CSase, EC 4.2.99.8) isozymes were purified from spinach leaves. Each isozyme was isolated to homogeneity by preparative PAGE. These isozymes were revealed to have different primary structures by amino-acid and proteinase digestion analyses, respectively. The enzymes designated as CSase 1, CSase 2 and CSase 3 with reference to the mobility on native PAGE were characterized with respect to physicochemical and enzymatic properties, and it was found that those enzymes had similar properties. It was also found that CSase 1 could be attributed to chloroplasts.

Improvement of the rectal bioavailability of latamoxef sodium by adjuvants following administration of a suppository.

The absorption of an antibiotic, latamoxef sodium (LMOX), following the rectal administration of a suppository containing adjuvants was investigated. A lipophilic base (Witepsol H15) was used. The rectal absorption of LMOX following the administration of a suppository without adjuvants was very low. Diclofenac sodium (DF) was used as an absorption promoter; it enhances rectal membrane permeability. The blood level of LMOX following the addition of DF(10 mg) to the base was increased only about 1.3-fold compared with that achieved with LMOX alone (difference not significant); even with a higher dose of DF, the absorption of LMOX was not sufficient. The release rate of LMOX from the base was slow. When Tween 80, a non-ionic surfactant, was added to improve the release rate of LMOX, the rate was sufficiently increased. The rectal absorption of LMOX on the addition of both Tween 80 and DF was markedly increased compared to that achieved with LMOX alone or with DF. These results indicate that the rectal absorption of LMOX after administration by a suppository was sufficiently improved by enhancing both the release rate from the base and the membrane permeability of the rectum. Lymphatic uptake and blood levels of LMOX were also investigated after the rectal administration of the LMOX preparation containing both Tween 80 and DF; the lymphatic uptake of LMOX was significantly enhanced compared with the LMOX preparation in which only DF was used as an adjuvant. The mechanism whereby adjuvants lead to the absorption of a non-absorbable drug, and the subsequent drug transportation routes through the membrane are discussed.

Gingival fluid IL-1 beta and IL-6 levels in menopause.

Menopause and oophorectomy without estrogen therapy (ED) have been associated with increased production of bone-active cytokines by peripheral blood mononuclear cells. The current study extended evaluation to gingival crevicular fluid (GCF) levels of interleukin (IL)-1 beta and IL-6 in such subjects compared to premenopausal and postmenopausal estrogen-treated females (ES). 13 ED and 13 ES Caucasians with a history of moderate-severe adult periodontitis provided GCF from 1-3 clinically identical sites each (5-6 mm probing depth, 5-7 mm clinical attachment loss, bleeding on probing). 30 s GCF samples were obtained and evaluated for IL-1 beta and IL-6 levels using two-site enzyme-linked immunosorbent assays (ELISAs). The frequency of GCF IL-1 beta-positive subjects was elevated in ED versus ES (92% versus 23%; p < 0.0004, chi 2 analysis). IL-6 was detected more frequently in ED subjects (23% versus 8%; not significant); however, the frequency of IL-6 detection was low in both groups due to short sampling times. These data support the concept that clinical conditions causing low estrogen environments allow increased local production of the bone-active cytokine IL-1 beta, and perhaps IL-6.