Impact of Cilostazol Pharmacokinetics on the Development of Cardiovascular Side Effects in Patients with Cerebral Infarction.

This study investigated the impact of polymorphisms of metabolic enzymes on plasma concentrations of cilostazol and its metabolites, and the influence of the plasma concentrations and polymorphisms on the cardiovascular side effects in 30 patients with cerebral infarction. Plasma concentrations of cilostazol and its active metabolites, and CYP3A5*3 and CYP2C19*2 and *3 genotypes were determined. The median plasma concentration/dose ratio of OPC-13213, an active metabolite by CYP3A5 and CYP2C19, was slightly higher and the median plasma concentration rate of cilostazol to OPC-13015, another active metabolite by CYP3A4, was significantly lower in CYP3A5*1 carriers than in *1 non-carriers (p = 0.082 and p = 0.002, respectively). The CYP2C19 genotype did not affect the pharmacokinetics of cilostazol. A correlation was observed between changes in pulse rate from the baseline and plasma concentrations of cilostazol (R = 0.539, p = 0.002), OPC-13015 (R = 0.396, p = 0.030) and OPC-13213 (R = 0.383, p = 0.037). A multiple regression model, consisting of factors of the plasma concentration of OPC-13015, levels of blood urea nitrogen, and pulse rate at the start of the therapy explained 55.5% of the interindividual variability of the changes in pulse rate. These results suggest that plasma concentrations of cilostazol and its metabolites are affected by CYP3A5 genotypes, and plasma concentration of OPC-13015, blood urea nitrogen, and pulse rate at the start of therapy may be predictive markers of cardiovascular side effects of cilostazol in patients with cerebral infarction.

Impact of Plasma Donepezil Concentration on Behavioral and Psychological Symptoms of Dementia in Patients with Alzheimer's Disease.

BACKGROUND/AIMS: The behavioral and psychological symptoms of dementia (BPSD) detract from the quality of life of not only dementia patients but also their family members and caregivers. Donepezil is used to treat Alzheimer's disease and is metabolized via cytochrome P450 (CYP) 2D6 and CYP3A4/5. It is controversial whether donepezil improves or exacerbates BPSD. This study investigated the relationships among BPSD, the pharmacokinetics of donepezil including its metabolite, 6-O-desmethyl donepezil, genetic polymorphisms of CYPs and P-glycoprotein, and patient backgrounds in 52 patients with Alzheimer's disease. METHODS: BPSD were assessed using the Neuropsychiatric Inventory (NPI), with scores ≥20 points defined as severe BPSD. Plasma donepezil and 6-O-desmethyl donepezil concentrations were measured using liquid chromatography-tandem mass spectrometry. RESULTS: Although significant relationships between NPI scores and plasma donepezil concentrations were not seen, none of the 15 patients (29%) with high plasma donepezil concentrations (≥60 ng/mL) developed severe BPSD. Polymorphisms of CYP2D6, CYP3A5, and ABCB1 did not influence NPI scores. There were no significant relationships between NPI and patient background factors such as dosing regimen, concomitant use of other drugs, or laboratory test results. Two patients who underwent multiple blood samplings over 2 years showed an inverse correlation between plasma donepezil concentrations and NPI scores. DISCUSSION/CONCLUSIONS: These results indicate that higher plasma concentrations of donepezil contribute to preventing or alleviating rather than developing or deteriorating BPSD.

Impact of CYP2D6, CYP3A5, and ABCB1 Polymorphisms on Plasma Concentrations of Donepezil and Its Metabolite in Patients With Alzheimer Disease.

BACKGROUND: Donepezil is one of the most commonly prescribed drugs for the treatment of Alzheimer disease. It is predominantly metabolized through CYP2D6 and to a lesser extent by CYP3A4/5. There are conflicting reports regarding the influence of CYP2D6, CYP3A5, and ABCB1 polymorphisms on the plasma concentration of donepezil. This study investigated the influence of these polymorphisms and sex on the plasma concentrations of donepezil and its active metabolite, 6-O-desmethyl donepezil (6ODD), in 47 patients with Alzheimer disease. METHODS: Plasma donepezil and 6ODD concentrations were measured using liquid chromatography tandem mass spectrometry. Sex, the concomitant use of psychotropics, and CYP2D6, CYP3A5, and ABCB1 polymorphisms were analyzed as possible influencers. RESULTS: The mean plasma concentrations of donepezil and 6ODD were well correlated (R2 = 0.418). The mean plasma concentration ratio of donepezil to 6ODD (metabolic ratio) was significantly lower in intermediate metabolizers of CYP2D6 than in extensive metabolizers. The metabolic ratio in patients receiving psychotropics was significantly lower than in those not receiving psychotropics. Among intermediate metabolizers, patients positive for CYP3A5 *3/*3 showed a significant increase in plasma mean 6ODD concentrations when compared with those who did not express this gene (CYP3A5 *1/*1 or *1/*3). CONCLUSIONS: Results indicate that the mean plasma concentration ratio of donepezil to 6ODD is associated with CYP2D6 polymorphism and the concomitant use of psychotropics in patients with Alzheimer disease. In intermediate metabolizers, CYP3A5 may play a significant role in the metabolism of donepezil.

Aged garlic extract ameliorates fatty liver and insulin resistance and improves the gut microbiota profile in a mouse model of insulin resistance.

Aged garlic extract (AGE) produced by the aging process has various beneficial pharmacological effects. In this study, the effects of AGE on fatty liver, insulin resistance and intestinal microbiota were compared between ddY-H mice, an insulin resistance mouse, and ddY-L mice, normal mice. Mice were fed an AGE-supplemented diet (4% w/w) for 7 weeks. The administration of AGE had no effect on the body weight and dietary intake of both types of mice. In the ddY-H mice, the serum levels of glucose and insulin were increased and glucose tolerance was impaired; however, the administration of AGE ameliorated these abnormal conditions. AGE did not have these effects in ddY-L mice. Triglyceride (TG) accumulation in the liver and fat absorption from the digestive tract were increased in the ddY-H mice; however, the administration of AGE reduced this increase. On the other hand, AGE exerted no such effects in the ddY-L mice. In addition, the gut microbiota has been shown to be closely associated with obesity, diabetes, dyslipidemia and non-alcoholic fatty liver disease in human and animal models. The bacterial composition of the gut microbiota in the feces of the ddY-H mice did not differ from that of the ddY-L mice at 5 weeks of age; however, it was altered in the mice at 9 and 12 weeks of age even when the mice were fed a standard diet. In the ddY-H mice, the relative presence of Lactobacillales was increased, while that of Bifidobacterium, Clostridium cluster XVIII and Prevotella was decreased. The alteration of the bacterial composition in the ddY-H mice was reversed by the administration of AGE; however, this effect of AGE was not observed in the ddY-L mice. On the whole, the findings of this study indicate that AGE improves abnormal fat accumulation and insulin resistance, and also alters the intestinal flora in ddY-H mice, suggesting the possibility that these effects of AGE may be related.

Therapeutic failure and eventual solution for skin necrosis and exposed tendon of the dorsum of the foot: A case report.

For the treatment of skin necrosis with exposed tendons in rheumatoid arthritis (RA) foot, we should perform microvascular free flap surgery at an early stage without conservative treatment considering the increased risk of infection and the decreased physical activity.

Prediction of neutrophil reduction using plasma paclitaxel concentration after administration in patients with uterine, ovarian, or cervical cancers in an outpatient clinic.

PURPOSE: Plasma paclitaxel (PTX) concentration 24 h or later after PTX administration may predict myelosuppression. Here, we explored predictive markers for neutropenia induced by intravenous administration of PTX in an outpatient clinic. METHODS: Thirty women suffering from uterine, ovarian or cervical cancer were enrolled in this study. PTX (mean dose: 167 mg/m2) was intravenously infused and followed by carboplatin. Plasma samples were obtained 4 h after PTX administration. Genotyping was carried out for CYP3A5*3, ABCB1 1236 C>T, 2677 G>T/A, and 3435 C>T. RESULTS: There was no significant relationship between genotype and reduced neutrophil count. Neutrophil reduction rate correlated with the patient's height, neutrophil count on the day of administration, and plasma PTX concentration. Multiple regression analysis with those three indices explained 47.7% of the interindividual variability of the neutrophil reduction rate. The model with plasma PTX concentration, patient's height, and plasma 6-α-hydroxy-paclitaxel /PTX concentration ratio also explained 30.0% of the interindividual variability for the neutrophil nadir count after PTX administration. CONCLUSIONS: These results indicate that neutrophil reduction after PTX administration can be partially predicted by multiple regression analysis involving plasma concentration data collected at outpatient clinics.

Determination of creatinine-related molecules in saliva by reversed-phase liquid chromatography with tandem mass spectrometry and the evaluation of hemodialysis in chronic kidney disease patients.

The serum concentrations of creatinine (Cre) and urea are used for the determination of the renal function. However, the use of blood is not always suitable due to the invasive, hygienic and infection problems during its sample collection and handling. In contrast, saliva is relatively clean and the samples can be quickly and noninvasively collected and easily stored. Therefore, the simultaneous determination of Arginine (Arg), creatine (Cr) and Cre in the saliva of chronic kidney disease (CKD) patients was performed by UPLC-ESI-MS/MS together with the saliva of healthy volunteers. The evaluation of hemodialysis of CKD patients was also carried out by the determinations before and after the dialysis. An HS-F5 column was used for the simultaneous determination of Arg, Cr and Cre in the saliva. These molecules were rapidly separated within 4 min and sensitively determined by the multiple reaction monitoring (MRM) of the precursor ion [M+H](+) â†’ product ions (m/z 175.1 â†’ 70.1 for Arg; m/z 132.0 â†’ 44.1 for Cr; m/z 114.0 â†’ 44.1 for Cre). The concentration of Cre in the CKD patients was higher than that in the healthy persons. The concentrations of Cre in the saliva of the patients before hemodialysis were moderately correlated with the serum Cre concentrations (R(2) = 0.661). Furthermore, the concentration in the saliva obviously decreased after hemodialysis (before 0.73 mg/dL, after 0.25 mg/dL; p < 0.02). Thus, the proposed detection method using saliva by UPLC-MS/MS is useful for the evaluation of the renal function in CKD patients. The present method offers a new option for monitoring the hemodialysis of CKD patients.

Age-Dependent Onset of Insulin Resistance in Insulin-Resistant Mice.

We have previously isolated spontaneous insulin-resistant mice (ddY-H) and non-insulin-resistant mice (ddY-L) from ddY mice. In the present study, age-dependent onset of insulin resistance in obese ddY-H mice was investigated by comparing with lean ddY-L mice. In ddY-H mice fed a standard diet, an increase in elevation of glucose-stimulated plasma insulin level, glucose intolerance in an intraperitoneal glucose tolerance test, and a reduction of hypoglycemic action of insulin were found at 9 weeks of age, but not at 6 weeks of age. When ddY-H mice were administered nateglinide, a greater elevation of plasma insulin level and a less decrease of serum glucose level were observed at 9 weeks of age. These changes developed progressively with age. These findings suggest that insulin resistance is induced at 9 weeks of age. The age-related change in insulin resistance was correlated with reductions in mRNA expression and protein content of the insulin receptor (InsR), and insulin receptor substrate (IRS)-1 and IRS-2 in the epididymal adipose tissue. On the other hand, in the liver, mRNA expression of InsR and IRS-1 did not change at any age, although that of the IRS-2 was reduced. Thus, in ddY-H mice, insulin resistance and glucose-stimulated hyper-secretion of insulin are induced at 9 weeks of age and are reciprocally affected, resulting in progression to a more severe state at an older age. Insulin resistance may be attributed, at least in part, to the decreases in the mRNA expressions and proteins of InsR, IRS-1 and IRS-2 in adipose tissue.

Posterior Tibial Artery Perforator-Based Fascial Flap for Skin Necrosis and Exposed Extensor Tendon After Revision Total Ankle Replacement.

CASE: Delayed wound-healing of anterior ankle incisions can be problematic for patients who have undergone total ankle replacement. We describe the case of a patient in whom a posterior tibial artery perforator-based fascial flap was effectively used to cover skin necrosis and to repair the extensor retinaculum in a wound following revision total ankle replacement. CONCLUSION: When a tendon is exposed in the ankle, a skin flap is generally required. The creation of a perforator-based fascial flap is a useful technique for covering a wound with an exposed tendon and is an alternative to a musculocutaneous flap.

Posterior Tibial Artery Perforator-Based Fascial Flap for Skin Necrosis and Exposed Extensor Tendon After Revision Total Ankle Replacement: A Case Report.

CASE: Delayed wound-healing of anterior ankle incisions can be problematic for patients who have undergone total ankle replacement. We describe the case of a patient in whom a posterior tibial artery perforator-based fascial flap was effectively used to cover skin necrosis and to repair the extensor retinaculum in a wound following revision total ankle replacement. CONCLUSION: When a tendon is exposed in the ankle, a skin flap is generally required. The creation of a perforator-based fascial flap is a useful technique for covering a wound with an exposed tendon and is an alternative to a musculocutaneous flap.

Effects of the inhibition of intestinal P-glycoprotein on aliskiren pharmacokinetics in cynomolgus monkeys.

Aliskiren is a substrate for P-glycoprotein (P-gp) and is metabolized via cytochrome P450 3A4 (CYP3A4). The aim of the present study was to assess whether P-gp influenced the pharmacokinetics of aliskiren and also if drug-drug interactions (DDIs) mediated through P-gp could be reproduced in cynomolgus monkeys. The study investigated the pharmacokinetics of aliskiren in mdr1a/1b gene-deficient (P-gp KO) and wild-type (WT) mice. The area under the plasma concentration-time curve (AUC) following the oral administration of aliskiren was 6.9-fold higher in P-gp KO mice than in WT mice, while no significant differences were observed in the AUC or total plasma clearance following the intravenous administration of aliskiren to P-gp KO mice. Then the pharmacokinetics of aliskiren were evaluated and DDIs between aliskiren and P-gp inhibitors, such as cyclosporin A (CsA) and zosuquidar, examined in cynomolgus monkeys. The AUC for aliskiren were 8.3- and 42.1-fold higher after the oral administration of aliskiren with the concomitant oral administration of zosuquidar and CsA at doses of 10 and 30 mg/kg, respectively. In contrast, the AUC after the intravenous and oral administration of aliskiren was not significantly affected by the oral administration of zosuquidar or intravenous administration of CsA, respectively. These results indicated that P-gp strictly limited the intestinal absorption of aliskiren in mice and monkeys, and also that the effects of intestinal P-gp inhibition by CsA or zosuquidar on the pharmacokinetics of aliskiren were sensitively reproduced in monkeys. In conclusion, aliskiren can be used as a sensitive substrate to evaluate intestinal P-gp inhibition in monkeys.

Profiling of chiral and achiral carboxylic acid metabolomics: synthesis and evaluation of triazine-type chiral derivatization reagents for carboxylic acids by LC-ESI-MS/MS and the application to saliva of healthy volunteers and diabetic patients.

Novel triazine-type chiral derivatization reagents, i.e., (S)-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidin-3-amine (DMT-3(S)-Apy) and (S)-4,6-dimethoxy-N-(pyrrolidin-3-yl)-1,3,5-triazin-2-amine (DMT-1(S)-Apy), were developed for the highly sensitive and selective detection of chiral carboxylic acids by UPLC-MS/MS analysis. Among the synthesized reagents, DMT-3(S)-Apy was a more efficient chiral reagent for the enantiomeric separation of chiral carboxylic acids in terms of separation efficiency by reversed-phase chromatography and detection sensitivity by ESI-MS/MS. The DMT-3(S)-Apy was used for the determination of 13 carboxylic acids in human saliva of healthy volunteers and diabetic patients. Various biological carboxylic acids including chiral carboxylic acids, and mono- and di-carboxylic acids were clearly identified in the saliva of healthy persons and diabetic patients. The concentrations of carboxylic acids detected in the saliva of diabetic patients were relatively higher than those in the healthy persons. Furthermore, the concentration of D-lactic acid (LA) and the ratio of D/L-LA in the diabetic patients were significantly higher than those in the healthy persons. The low ratio of D/L-LA in healthy persons was also identified to be independent of age and sex. These results suggest that the determination of the D/L-LA ratio in saliva might be applicable for the diagnosis of diabetes. Based on these observations, DMT-3(S)-Apy seems to be a useful chiral derivatization reagent for the determination not only of chiral carboxylic acids but also achiral ones. In conclusion, the proposed method using DMT-3(S)-Apy is useful for the carboxylic acid metabolomics study of various specimens.

Determination of acetone in saliva by reversed-phase liquid chromatography with fluorescence detection and the monitoring of diabetes mellitus patients with ketoacidosis.

BACKGROUND: In diabetes mellitus (DM) patients with ketoacidosis, ketone bodies, i.e., acetone, acetoacetic acid (AA) and ß-hydroxybutyric acid (HA), are increased in the blood and urine. Acetone is also excreted by breathing due to the spontaneous decomposition of AA. Thus, the increase in acetone has been considered as one of the biomarkers for the diagnosis of DM. However, the determination of acetone in one's breath is not recommended because of the sample handling difficulty. We measured acetone in saliva by reversed-phase liquid chromatography (LC) with fluorescence (FL) detection. The proposed method was applied to the determination of acetone in the saliva of healthy volunteers and DM patients with and without ketoacidosis. METHODS: 3-Pentanone (I.S.) and DBD-H in acetonitrile were added to freshly collected saliva and reacted at room temperature for 20 min in the presence of trifluoroacetic acid. After the reaction, the solution was centrifuged at 10,000 × g and 4 °C for 5 min. The supernatant was separated by reversed-phase LC and the FL detected at 550 nm (excitation at 460 nm). RESULTS: The concentrations of acetone in the DM patients with ketoacidosis were significantly higher than those of the normal subjects and DM patients without ketoacidosis. Furthermore, the total contents of the ketone bodies in the blood correlated with acetone in the saliva of the DM patients. The concentrations of acetone in the saliva of an emergency patient also correlated with the ketone bodies in the blood at each sampling time. CONCLUSION: The proposed method using LC-FL seems to be useful for the determination of acetone in the saliva of DM patients with ketoacidosis. The method offers a new option for the diagnosis and monitoring of DM patients with ketoacidosis.

Hepatic steatosis with relation to increased expression of peroxisome proliferator-activated receptor-γ in insulin resistant mice.

We have isolated insulin resistant mice (ddY-H mice) which are spontaneously induced even if fed with the standard chow pellets. Since marked accumulation of triglycerides (TG) in liver was observed, the present study investigated causes of hepatic TG accumulation in ddY-H mice fed with the standard chow pellets. In ddY-H mice, hepatic TG content increased from seven-weeks of age, and further marked accumulation of TG was observed at 20-weeks of age. Histologically, fat droplets appeared in pericentral parenchymal cells of the liver from nine-weeks of age, and the size and number of droplets were increased in hepatic lobules at 15-weeks of age, suggesting hepatic steatosis was spontaneously induced. Although secretion of TG from liver to blood in ddY-H mice was not increased, fat absorption from the digestive tract was significantly enhanced. The mRNA expressions of peroxisome proliferator-activated receptor γ (PPARγ) involved in fat accumulation and fatty acid translocase (CD36) involved in the transportation of fatty acid into the liver were markedly increased. However, gene expressions of factors involved in lipogenesis, ß-oxidation of fatty acid and lipoprotein secretion were not changed. Pioglitazone (9 mg/kg), the PPARγ agonist, administered for six weeks deteriorated hepatic steatosis in ddY-H mice. Although pioglitazone did not affect gene expressions of PPARγ in the liver, CD36 and fat-specific protein 27 (fsp27), targets of PPARγ, were markedly elevated. These results suggest that, in the livers of ddY-H mice, hepatic steatosis is induced by increased incorporation of fatty acid into the liver via increased PPARγ expression.

Infiltration of M1 macrophages into adipose tissue of ddY-H mice preceding spontaneous appearances of insulin resistance.

We have isolated insulin resistant mice (ddY-H mice) which are spontaneously induced at 12-weeks of age even if fed with the standard chow pellets. Since accumulated evidences have suggested that an appearance of insulin resistance is associated with obesity and a state of inflammation in adipose tissue, the present study investigated an appearance of macrophages in adipose tissue of ddY-H mice. Although ddY-H mice were fed the standard chow pellets ad libitam, increases in body weight, adipose tissue mass, and fat cell size were observed. In adipose tissues of ddY-H mice, gene expression of monocyte chemoattractant protein-1 (MCP-1) elevated slightly at 5-weeks of age and was maintained at higher levels at 9- and 12-weeks of age, and MCP-1 content in adipose tissue increased 2-fold at 12-weeks of age. Also, increased gene expressions of CD68 and F4/80, markers of macrophage, in adipose tissue were observed at 9-weeks of age. In addition, F4/80 positive cells were histologically found in adipose tissue at 15-weeks of age but not at 7-weeks of age, suggesting an increased infiltration of macrophage into adipose tissue. In adipose tissue of ddY-H mice, gene expressions of CD11c and toll-like receptor 4 (TLR4), markers of proinflammatory macrophages (M1), markedly increased although those of CD163 and mannose receptor (MR), markers of anti-inflammatory macrophages (M2), did not change. These results suggest that proinflammatory (M1) macrophages infiltrate into enlarged adipose tissues of ddY-H mice, which is preceding spontaneous appearance of insulin resistance.

Influence of the slow infusion of a soybean oil emulsion on plasma cytokines and ex vivo T cell proliferation after an esophagectomy.

BACKGROUND: Lipid emulsions have been suggested to reduce immune responses, particularly in severely stressed patients. The authors investigated the influence of the slow intravenous infusion of a soybean oil-based lipid emulsion on some immune parameters in patients who had undergone an esophagectomy for esophageal cancer. METHODS: Thirty-two patients who had undergone an esophagectomy were randomly divided into a lipid emulsion (LPD)-treated group and a control group. All patients received parenteral feeding with a glucose-based solution. Patients in the LPD group received 100 mL of a 20% soybean oil emulsion for 7 days after the esophagectomy in addition to the glucose-based feeding. A slow infusion rate (0.09-0.12 g/kg/h) was adopted to take account of the intrinsic degradation of infused lipids. Immune responses were measured based on lymphocyte proliferation and serum concentrations of monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). The authors also measured levels of rapid turnover proteins (ie, transferrin, prealbumin, and retinol-binding protein). RESULTS: Phytohemagglutinin- and concanavalin A-stimulated lymphocyte proliferation significantly decreased after the esophagectomy, but no significant difference was seen between the LPD and control groups. No significant difference in changes in plasma concentrations of MCP-1, IL-6 and TNF-α occurred between the 2 groups either. Plasma concentrations of rapid turnover proteins did not differ between the groups. CONCLUSIONS: These results indicate that the lipid emulsion did not affect the immune parameters measured in patients who had undergone an esophagectomy when administered at a slow rate.

Simultaneous determination of DL-lactic acid and DL-3-hydroxybutyric acid enantiomers in saliva of diabetes mellitus patients by high-throughput LC-ESI-MS/MS.

A simultaneous determination method for the enantiomers of chiral carboxylic acids by the combination of ultraperformance liquid chromatography and mass spectrometry (UPLC-MS/MS) has been developed. (S)(+)-1-(2-Pyrrolidinylmethyl)-pyrrolidine (S-PMP) was used as the derivatization reagent for the high-throughput determination of biological chiral carboxylic acids, i.e., lactic acid (LA) and 3-hydroxybutyric acid (HA). The S-PMP efficiently reacted with the carboxylic acids under mild conditions at room temperature in the presence of 2,2'-dipyridyl disulfide and triphenylphosphine. The resulting S-PMP derivatives were highly responsive in the electrospray ionization (ESI)-MS operating in the positive-ion mode and gave characteristic product ions during the MS/MS, which enabled the sensitive detection using selected reaction monitoring. The derivatization was effective for the enantiomeric separation of the chiral carboxylic acids, and the resolution values of DL-LA and DL-HA were 4.91 and 9.37, respectively. Furthermore, a rapid separation of the derivatives of DL-LA and DL-HA within 7 min was performed using the UPLC system. The limits of detection on the column were in the low femtogram range (5-12 fg). The proposed procedure was successfully applied for the determination of the D- and L-isomers of LA and HA in the saliva of diabetes mellitus (DM) patients and healthy volunteers. The D-LA in DM patients was clearly higher than that in normal subjects. The derivatization followed by UPLC-ESI-MS/MS enabled the enantiomeric separation and detection of trace amounts of LA and HA in human saliva with a simple pretreatment and small sample volume.

Biomarker discovery in biological specimens (plasma, hair, liver and kidney) of diabetic mice based upon metabolite profiling using ultra-performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry.

BACKGROUND: The number of diabetic patients has recently been increasing worldwide. Diabetes is a multifactorial disorder based on environmental factors and genetic background. In many cases, diabetes is asymptomatic for a long period and the patient is not aware of the disease. Therefore, the potential biomarker(s), leading to the early detection and/or prevention of diabetes mellitus, are strongly required. However, the diagnosis of the prediabetic state in humans is a very difficult issue, because the lifestyle is variable in each person. Although the development of a diagnosis method in humans is the goal of our research, the extraction and structural identification of biomarker candidates in several biological specimens (i.e., plasma, hair, liver and kidney) of ddY strain mice, which undergo naturally occurring diabetes along with aging, were carried out based upon a metabolite profiling study. METHODS: The low-molecular-mass compounds including metabolites in the biological specimens of diabetic mice (ddY-H) and normal mice (ddY-L) were globally separated by ultra-performance liquid chromatography (UPLC) using different reversed-phase columns (i.e., T3-C18 and HS-F5) and detected by electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). The biomarker candidates related to diabetes mellitus were extracted from a multivariate statistical analysis, such as an orthogonal partial least-squares-discriminant analysis (OPLS-DA), followed by a database search, such as ChemSpider, KEGG and HMDB. RESULTS: Many metabolites and unknown compounds in each biological specimen were detected as the biomarker candidates related to diabetic mellitus. Among them, the elucidation of the chemical structures of several possible metabolites, including more than two biological specimens, was carried out along with the comparison of the tandem MS/MS analyses using authentic compounds. One metabolite was clearly identified as N-acetyl-L-leucine based upon the MS/MS spectra and the retention time on the chromatograms. CONCLUSIONS: N-acetyl-L-leucine is an endogenous compound included in all biological specimens (plasma, hair, liver and kidney). Therefore, this metabolite appears to be a potential biomarker candidate related to diabetes. Although the structures of other biomarker candidates have still not yet determined, the present approach based upon a metabolite profiling study using UPLC-ESI-TOF-MS could be helpful for understanding the abnormal state of various diseases.

A new diabetic mouse model derived from the ddY strain.

Selective breeding was used to isolate hyperglycemic (ddY-H) or normoglycemic (ddY-L) mice that had been induced by fasting and refeeding. Serum glucose levels 12 h after 48 h-fasting were high in male ddY-H mice, but relatively low in male ddY-L mice compared with control ddY mice. Glucose tolerance was impaired in ddY-H mice maintained with standard chow pellets ad libitum at 12 weeks of age, and serum glucose and insulin levels were significantly increased after overnight fasting at 15 weeks of age. ddY-L mice indices did not differ from ddY mice indices, suggesting that insulin resistance is spontaneously induced in ddY-H mice. Increases in urinary excretion and urinary sugar accompanied by increased body mass were observed in all ddY-H mice, but not in ddY or ddY-L mice, at 27 weeks of age, indicating the induction of diabetic symptoms. Cross-mating between ddY-H and ddY-L mice was used to certify the genetic involvement in impaired glucose tolerance. This was not induced in mice born from male ddY-H and female ddY-L mice, or from female ddY-H and male ddY-L mice. In conclusion, ddY-H mice are a useful diabetic mouse model that show spontaneously induced insulin resistance followed by diabetic symptoms that are maintained by standard chow pellets. Their characteristics are recessively inherited, and ddY-L mice are an appropriate choice as control mice.

Botulinum toxin type A in post-stroke upper limb spasticity.

OBJECTIVE: To evaluate the efficacy and safety of one-time injections of botulinum toxin type A (BoNTA) in Japanese patients with post-stroke upper limb spasticity. RESEARCH DESIGN AND METHODS: In a multicentre, randomised, double-blind, parallel-group, placebo-controlled study, 109 patients with upper limb spasticity were randomised to receive a single treatment with lower-dose (120-150 U) or higher-dose (200-240 U) BoNTA or placebo into upper limb muscles. CLINICAL TRIAL REGISTRATION: NCT00460564. MAIN OUTCOME MEASURES: The tone of the wrist flexor was assessed at baseline and at weeks 0, 1, 4, 6, 8 and 12 using the Modified Ashworth Scale (MAS) for wrist, finger, thumb and disability in activities of daily living (ADL) was rated using the 4-point Disability Assessment Scale (DAS). The primary endpoint was area under the curve (AUC) of the change from baseline in the MAS wrist score in the higher-dose groups. RESULTS: Subjects were randomised with 51 in the higher BoNTA group, 26 in the higher-dose placebo group, 21 in the lower BoNTA group and 11 in the lower-dose placebo group. Significant improvement in spasticity with higher-dose BoNTA was demonstrated by a mean difference in the AUC of the change from baseline in the MAS wrist score between the higher-dose BoNTA group and the higher-dose placebo group of -6.830 (p < 0.001, t-test), no significant different was demonstrated between the lower-dose BoNTA group and the lower-dose placebo group (p = 0.215, t-test). Significant improvements with higher-dose BoNTA were also observed in the DAS scores for limb position (p = 0.001-0.022) at all time points and dressing (p = 0.018-0.038, Wilcoxon test) at weeks 6, 8 and 12. No clinically relevant difference was noted in the frequency of treatment-related adverse events between BoNTA-treated and placebo-treated patients. The long-term efficacy and safety, and the effects on rehabilitation of BoNTA on upper limb will be evaluated using the data obtained in the open-label phase. CONCLUSIONS: Higher-dose BoNTA reduced spasticity in upper limb muscles and improved ADL performance in terms of limb position and dressing. BoNTA is safe and effective in the treatment of post-stroke upper limb spasticity.