Human nails metabolite analysis: A rapid and simple method for quantification of uric acid in human fingernail by high-performance liquid chromatography with UV-detection.

A rapid and simple analytical method for the quantification of uric acid (UA) in human fingernails by high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection is described. UA was extracted from human fingernail samples at 90°C for 20min, then separated on an Inertsil ODS-2 column (250×4.6mm I.D., 5.0µm, GL Sciences) by isocratic elution using methanol: 74mM phosphate buffer (pH 2.2) 2:98 (v/v). An UV detector was used to monitor at 284nm. The results indicated that under optimized measurement conditions results were achieved within 8.0min, and a good linearity was achieved from the calibration curves (r(2)>0.9999) in the range of 1.0-10000ng; the limit of detection (S/N=3) was 2.0pg, the inter-day and intra-day assay precisions were all less than 0.46% and the mean recoveries (%) of the uric acid spiked in the human fingernail were 101.95%. The amounts of UA in the fingernails of healthy volunteers were determined.

First observation of N-acetyl leucine and N-acetyl isoleucine in diabetic patient hair and quantitative analysis by UPLC-ESI-MS/MS.

BACKGROUND: Type 2 diabetes patients (DP) have significantly higher plasma levels of valine, leucine, isoleucine and alanine than the controls. Specific amino acids may acutely and chronically regulate insulin secretion from the pancreatic ß-cells. We recently identified a metabolic signature of N-acetyl leucine (Ac-Leu) that strongly predicts diabetes development in mice hair. The Ac-Leu appears to be a potential biomarker candidate related to diabetes. However, the determination of Ac-Leu in human hair has not been reported. We measured the Ac-Leu, and its structure is similar to N-acetyl isoleucine (Ac-Ile) in human hair by ultra-performance liquid chromatography (UPLC) with electrospray ionization tandem mass spectrometry (ESI-MS/MS). The developed method was applied to the determination of Ac-Leu and Ac-Ile in the hair of healthy volunteers (HV) and DP. METHODS: Ac-Leu, Ac-Ile and N-acetyl norleucine (Ac-Nle, IS) were extracted from human hair samples by a micropulverized extraction procedure, then separated on a C18 column by isocratic elution of acetonitrile-0.1% formic acid in water:0.1% formic acid (14:86, vol./vol.). MRM using the fragmentation transitions of m/z 174.1→86.1 in the positive ESI mode was performed to quantify the N-acetyl leucine, N-acetyl isoleucine and IS. RESULTS: Ac-Leu, Ac-Ile and Ac-Nle in the human hair samples were completely separated by isocratic elution of a 5.0 min duration wash program using a reversed-phase column, and sensitively detected by LC-MS/MS in the ESI(+) MRM mode. The amounts of Ac-Leu and Ac-Ile in the hairs of HV and DP were determined. When comparing the concentrations between DP and those from HV, a statistically significant correlation was observed for the Ac-Leu (p<0.001) and Ac-Ile (p<0.01). CONCLUSIONS: The proposed method is useful for the determination of Ac-Leu and Ac-Ile in the hairs of DP and HV. Human hair may serve as a noninvasive biosample for the diagnosis of diabetes.

A quantitative analysis of the polyamine in lung cancer patient fingernails by LC-ESI-MS/MS.

A quantitative analysis of polyamines in lung cancer patient fingernails by the combination of 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole derivatives and liquid chromatography-electrospray ionization tandem mass spectrometry is described. The reaction of the reagent with eight kinds of polyamines, that is, N(1) -acetylputrescine (N(1) -actPUT), N(8) -acetylspermidine, N(1) -acetylspermine, 1,3-diaminopropane, putrescine (PUT), cadaverine, spermidine and spermine (SPM) effectively occurs at 60 °C for 30 min. The detection limits (signal-to-noise ratio 5) were 5-100 fmol. A good linearity was achieved from the calibration curves, which was obtained by plotting the peak area ratios of the analytes relative to the internal standard (IS), that is, 1,6-diaminohexane, vs the injected amounts of polyamines (r(2) > 0.996), and the intra-day and inter-day assay precisions were <9.84%. Furthermore, the recoveries (%) of the polyamines spiked in the human fingernails were 89.14-110.64. The present method was applied to human fingernail samples from 17 lung cancer patients and 39 healthy volunteers. The polyamine concentration was different based on the gender, that is, the N(1) -actPUT and PUT contents were 3.10 times and 2.56 times higher in healthy men than in women, respectively. Additionally, in the lung cancer patient group, as compared with the healthy volunteers, the concentrations of SPM had a statistically significant (p < 0.05) correlation. Therefore, because the proposed method provides a good mass accuracy and the trace detection of the polyamines in human fingernails, this analytical technique could be a noninvasive technique to assist in the diagnosis and assessment of disease activity in lung cancer patients.

Rapid and sensitive determination of diacetylpolyamines in human fingernail by ultraperformance liquid chromatography coupled with electrospray ionization tandem mass spectrometry.

A rapid and sensitive ultraperformance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) method has been developed and validated for quantitatively determining diacetylpolyamines in the human fingernail. N(1),N(8)-diacetylspermidine (DiAct-Spd), N(1),N(12)- diacetylspermine (DiAct-Spm) and 1,6-diaminohexane (DAH) the [internal standard (IS)] were extracted from human fingernail samples by MeOH: 5 M HCl solution, followed by 4-(N,N-dimethylaminosulfonyl)-7-fluoro- 2,1,3-benzoxadiazole (DBD-F) derivatization, and then separated on an ACQUITY BEH C18 column with a gradient elution of acetonitrile and water containing 0.1% formic acid. The derivatives of the diacetylpolyamines were fully separated within a short run time (3.0 min). The triple quadrupole mass spectrometric detection was performed in the multiple reactions monitoring (MRM) mode by the UPLC-ESI- MS/MS system in the positive ionization mode. MRM using the fragmentation transitions of m/z 455.20→ 100.07, 737.25 → 100.07 and 567.10 → 479.07 in the positive ESI mode was performed to quantify DiAct-Spd, DiAct-Spm and IS, respectively. The calibration curve is between 0.04 ng mL(-1) for DiAct-Spd and DiAct-Spm. The detection limits (signal to noise ratio of five) were 5-10 pg mL(-1). A good linearity was achieved from the calibration curves (r(2) >0.9999), and the intra-day and inter-day assay precisions were less than 7.06%. Furthermore, the recoveries (%) of the diacetylpolyamines spiked in the human fingernails were 79.18-97.11. The present method proved that the high sensitivity is characterized by the specificity and feasibility of the sample analysis. Consequently, the proposed method was used to analyze human fingernail samples from 15 lung- cancer patients and 22 healthy volunteers. Diacetylpolyamines were detected from the fingernails of the lung- cancer patients for the first time. The concentration of DiAct-Spd in the lung-cancer patient group tended to be higher than those in the healthy volunteers.

Yangonin blocks tumor necrosis factor-α-induced nuclear factor-κB-dependent transcription by inhibiting the transactivation potential of the RelA/p65 subunit.

The nuclear factor-κB (NF-κB) transcription factors control many physiological processes including inflammation, immunity, and apoptosis. In our search for NF-κB inhibitors from natural resources, we identified yangonin from Piper methysticum as an inhibitor of NF-κB activation. In the present study, we demonstrate that yangonin potently inhibits NF-κB activation through suppression of the transcriptional activity of the RelA/p65 subunit of NF-κB. This compound significantly inhibited the induced expression of the NF-κB-reporter gene. However, this compound did not interfere with tumor necrosis factor-α (TNF-α)-induced inhibitor of κBα (IκBα) degradation, p65 nuclear translocation, and DNA-binding activity of NF-κB. Further analysis revealed that yangonin inhibited not only the induced NF-κB activation by overexpression of RelA/p65, but also transactivation activity of RelA/p65. Moreover, yangonin did not inhibit TNF-α-induced activation of p38, but it significantly impaired activation of extracellular signal-regulated kinase 1/2 and stress-activated protein kinase/c-Jun NH(2)-terminal kinase. We also demonstrated that pretreatment of cells with this compound prevented TNF-α-induced expression of NF-κB target genes, such as interleukin 6, interleukin 8, monocyte chemotactic protein 1, cyclooxygenase-2 and inducible nitric oxide. Taken together, yangonin could be a valuable candidate for the intervention of NF-κB-dependent pathological conditions such as inflammation.

The protective effects of total saponins from Ornithogalum saundersiae (Liliaceae) on acute hepatic failure induced by lipopolysaccharide and D-galactosamine in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: This study examined the protective effects of total saponins from Ornithogalum saundersiae (Liliaceae) on D-galactosamine (D-GalN) and lipopolysaccharide (LPS)-induced fulminant hepatic failure. MATERIALS AND METHODS: Total saponins of Ornithogalum saundersiae (Liliaceae) (OC) were prepared with ethyl alcohol extract from bulbs of the plant. Mice were given an intraperitoneal injection of D-GalN (700 mg/kg)/LPS (10 µg/kg). OC (100 mg/kg, 200 mg/kg and 300 mg/kg) was administered orally for 3 days continuously, and at the last day at 1 h before the D-GalN/LPS injection. Mice were sacrificed at 8 h after the D-GalN/LPS injection. The liver injury was assessed biochemically, investigating aspartate aminotransferase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA), glutathione (GSH) activities, and the expressions of caspase-3 and hypoxia inducible factor-1α (HIF-1α) as well. Tumor necrosis factor (TNF-α) content was measured after D-GalN/LPS induced 1 h by ELISA assay. The survival rates after application of OC in 24 h also were observed. RESULTS: D-GalN/LPS increased the serum aminotransferase levels and lipid peroxidation, while decreased the reduced glutathione level. The pretreatment with OC attenuated these changes in a dose-dependent manner. Elevation of TNF-α level and activation of caspase-3, HIF-1α were observed in the D-GalN/LPS group, which was attenuated by OC. The survival rate of the OC groups was significantly higher than that of the D-GalN/LPS group. CONCLUSIONS: Protection afforded by OC against D-GalN/LPS-induced fulminant hepatic failure is the result of reduced oxidative stress, inhibited expression of caspase-3, HIF-1α, and anti-apoptotic activity.

Gentiana manshurica Kitagawa prevents acetaminophen-induced acute hepatic injury in mice via inhibiting JNK/ERK MAPK pathway.

AIM: To investigate the in vivo hepatoprotective effects and mechanisms of Gentiana manshurica Kitagawa (GM) in acetaminophen (APAP)-induced liver injury in mice. METHODS: GM (200, 150 or 50 mg/kg body weight) or N-acetyl-L-cysteine (NAC; 300 mg/kg body weight) was administrated orally with a single dose 2 h prior to APAP (300 mg/kg body weight) injection in mice. RESULTS: APAP treatment significantly depleted hepatic glutathione (GSH), increased serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) and malonyldialdehyde (MDA) and 4-hydroxynonenal levels, and decreased hepatic activity of glutathione peroxidase (GSH-px) and superoxide dismutase (SOD). However, the pretreatment of GM significantly alleviated APAP-induced oxidative stress by increasing GSH content, decreasing serum ALT, AST and MDA, and retaining the activity of GSH-px and SOD in the liver. Furthermore, GM pretreatment can inhibit caspase-3 activation and phosphorylation of c-Jun-NH2-terminal protein kinase 2 (JNK1/2) and extracellular signal-regulated kinase (ERK). GM also remarkably attenuated hepatocyte apoptosis confirmed by the terminal deoxynucleotidyl transferase mediated dUTP nick end-labeling method. CONCLUSION: Hepatoprotective effects of GM against APAP-induced acute toxicity are mediated either by preventing the decline of hepatic antioxidant status or its direct anti-apoptosis capacity. These results support that GM is a potent hepatoprotective agent.

Acanthoic acid, a diterpene in Acanthopanax koreanum, protects acetaminophen-induced hepatic toxicity in mice.

The protective effect of a diterpenoid acanthoic acid (AA) isolated from Acanthopanax koreanum Nakai was investigated in acetaminophen (APAP)-induced hepatic toxicity. Drug-induced hepatotoxicity induced by an intraperitoneal (i.p.) injection of 300mg/kg (sub-lethal dose) of APAP. Pretreatment with AA (50 and 100mg/kg) orally 2h before the APAP administration attenuated the APAP-induced acute increase in serum aspartate aminotransferase (AST), and alanine aminotransferase (ALT) activites, replenished the depleted hepatic glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) activities, decreased malondialdehyde (MDA) level and considerably reduced the histopathological alterations in a manner similar to silymarin (Sily). Immunohistochemical analyses also demonstrated that AA could reduce the appearance of necrosis regions as well as caspase-3 and hypoxia inducible factor-1alpha (HIF-1alpha) expression in liver tissue. Our results indicated that AA protected liver tissue from the oxidative stress elicites by APAP-induced liver damage and suggestes that the hepatic protection mechanism of AA would relate to antioxidation and hypoxia factor on APAP-induced hepatotoxicity.

Hepatoprotective effects of salidroside on fulminant hepatic failure induced by D-galactosamine and lipopolysaccharide in mice.

OBJECTIVES: The aim was to investigate the protective effect of salidroside isolated from Rhodiola sachalinensis A. Bor. (Crassulaceae) on D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure. METHODS: Hepatotoxicity was induced by an intraperitoneal injection of D-galactosamine (700 mg/kg) and lipopolysaccharide (10 mug/kg); salidroside (20, 50 and 100 mg/kg) was administered intraperitoneally 1 h before induction of hepatoxicity. Liver injury was assessed biochemically and histologically. KEY FINDINGS: Salidroside attenuated the induced acute increase in serum aspartate aminotransferase and alanine aminotransferase activities, and levels of tumour necrosis factor-alpha levels and serum nitric oxide. It restored depleted hepatic glutathione, superoxide dismutase, catalase and glutathione peroxidase activities, decreased malondialdehyde levels and considerably reduced histopathological changes. Histopathological, immunohistochemical and Western blot analyses also demonstrated that salidroside could reduce the appearance of necrotic regions and expression of caspase-3 and hypoxia-inducible factor-1alpha in liver tissue. CONCLUSIONS: Salidroside protected liver tissue from the oxidative stress elicited by D-galactosamine and lipopolysaccharide. The hepatoprotective mechanism of salidroside appear to be related to antioxidant activity and inhibition of hypoxia-inducible factor-1alpha.

A diterpenoid acanthoic acid from Acanthopanax koreanum protects against D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure in mice.

The hepatoprotective effects of a diterpenoid acanthoic acid isolated from Acanthopanax koreanum NAKAI were evaluated in a D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure mouse model. Mice were pretreated orally with acanthoic acid 12 and 1 h before intraperitoneal injection of D-galactosamine and lipopolysaccharide. Pretreatment with the compound markedly reduced lethal liver injury in experimental animals. The effects were likely associated with a significant decrease in serum tumor necrosis factor (TNF)-alpha levels, which are correlated not only with those of alanine aminotransferase and aspartate aminotransferase but also with the reduced number of apoptotic hepatocytes in the liver as confirmed using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling method and DNA fragmentation assay. These results suggest that acanthoic acid protects against D-galactosamine/lipopolysaccharide-induced fulminant liver failure at least in part by a mechanism associated with the down-regulation of TNF-alpha secretion.

Protective effect of selenium-enriched Lactobacillus on CCl4-induced liver injury in mice and its possible mechanisms.

AIM: To study the protective effects and mechanisms of Se-enriched lactobacillus on liver injury caused by carbon tetrachloride (CCl4) in mice. METHODS: Seventy-two ICR mice were randomly divided into four groups: normal group, CCl4-induced model group, low Se-enriched lactobacillus treatment group (L-Se group), and high Se-enriched lactobacillus treatment group (H-Se group). During a 3-wk experimental period, the common complete diet was orally provided daily for normal group and model group, and the mice in L-Se and H-Se groups were given a diet with 2 and 4 mg of organoselenium from Se-enriched lactobacillus per kg feed, respectively. From the 2nd wk of experiment, the model group, L-Se group, and H-Se group received abdominal cavity injection of olive oil solution containing 500 mL/L CCl4 (0.07 mL/100 g body mass) to induce liver injury, and the normal group was given olive oil on every other day for over 2 wk. In the first 2 wk post injection with CCl4, mice in each group were killed. The specimens of blood, liver tissue, and macrophages in abdominal cavity fluid were taken. Then the activities of the following liver tissue injury-associated enzymes including glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) as well as malondialdehyde (MDA) content were assayed. Changes of phagocytic rate and phagocytic index in macrophages were observed with Wright-Giemsa stain. Plasma TNF-alpha level was measured by radioimmunoassay. The level of intracellular free Ca2+ ([Ca2+]i) in hepatocytes was detected under a laser scanning confocal microscope. RESULTS: During the entire experimental period, the AST and ALT activities in liver were greatly enhanced by CCl4 and completely blunted by both low and high doses of Se-enriched lactobacillus. The Se-enriched lactobacillus-protected liver homogenate GSH-Px and SOD activities were higher or significantly higher than those in model group and were close to those in normal group. CCl4 significantly increased MDA content in liver homogenates, while administration of Se-enriched lactobacillus prevented MDA elevation. Phagocytic rate and phagocytic index of macro-phages decreased after CCl4 treatment compared to those in normal control, but they were dramatically rescued by Se-enriched lactobacillus, showing a greatly higher phagocytic function compared to model group. CCl4 could significantly elevate plasma TNF-alpha and hepatocyte [Ca2+]i level, which were also obviously prevented by Se-enriched lactobacillus. CONCLUSION: Se-enriched lactobacillus can intervene in CCl4-induced liver injury in mice by enhancing macrophage function activity to keep normal and beneficial effects, elevating antioxidant-enzyme activities and reducing lipid peroxidation reaction, inhibiting excessive release of TNF-alpha, preventing the dramatic elevation of [Ca2+]i in hepatocytes.

Action of NO and TNF-alpha release of rats with cadmium loading in malfunctiion of multiple system organ.

Thirty-six healthy Sprague-Dawley male rats were used and divided randomly into a control group (group C), a medium-dose cadmium loading group (group M) and a high-dose cadmium loading group (group H). Cadmium chloride was diluted with saline to contain 0.4 mg/ml of autoclaved cadmium solution. Groups M and H were injected in the abdominal cavity with 0.5 and 1.0 mg of cadmium per kg body weight respectively, and group C with saline of the same dosage as in group H over 7 d. Six rats of each group were killed on the 4th day and 7th day after cadmium loading, respectively, and blood, testis, liver and heart were collected. Cadmium content, changes in nitric oxide and tumor necrosis factor-alpha were studied in blood and the tissues of testis, liver and heart. Results showed that during the entire experimental period, body weight in groups M and H decreased significantly as compared with that in group C; cadmium concentration increased significantly in testis, heart and liver of groups M and H, and rose with increased dosage and time of cadmium loading; there was no obvious difference in plasma nitric oxide between groups M and C, though nitric oxide was higher in group M than in group C. Nitric oxide in group H was significantly superior to that in group C. Plasma tumor necrosis factor-alpha was markedly higher in groups M and H than in group C. Nitric oxide contents in the rat s testis, liver and heart homogenates with cadmium loading were higher than those in group C or significantly superior to group C. The same changes in tumor necrosis factor-alpha in the tissue homogenates of the testis and heart were found, but no obvious difference in tumor necrosis factor-alpha in the liver between the three groups was observed. It is suggested that the massive release of nitric oxide and tumor necrosis factor-alpha induced by cadmium loading may play an important role in the induction of malfunction of multiple systems or organs in rats.

Protective effect of Rhodiola sachalinensis extract on carbon tetrachloride-induced liver injury in rats.

This study was carried out to investigate the protective effect of an aqueous extract from the root of Rhodiola sachalinensis (RSE) on liver injury induced by repetitive administration of carbon tetrachloride in rats. RSE was given orally to rats at doses of 50, 100 or 200 mg/kg throughout the carbon tetrachloride treatment for 28 days. In rats treated with carbon tetrachloride, the levels of hydroxyproline and malondialdehyde (MDA) in the liver, and serum enzyme activities were significantly increased. RSE treatment significantly reduced the levels of liver hydroxyproline and MDA, and serum enzyme activities, in accordance with improved histological findings. Immunohistological findings indicated RSE treatment inhibited hepatic stellate cell activation, which is a major step for collagen accumulation during liver injury. These data suggest that RSE protects the liver from repetitive injury induced by carbon tetrachloride in rats.

[Effects of chronic cadmium loading on the testis and endocrine function of reproduction in male rats].

Sixty healthy Sprague-Dawley male rats were used and divided randomly into control group (group C), cadmium loading group with medium dose (group M) and cadmium loading group with high dose (group H). Groups C, M and H were orally dosed daily with 0, 5 and 10 mg/kg of cadmium for over 6 weeks. Effects of cadmium loading on testis and endocrine function of reproduction in male rats were studied. The results showed that the zinc content decreased slightly in testis and plasma, and the cadmium concentration increased significantly in the testis of groups M and H; while the plasma levels of cadmium and zinc had no obvious difference as compared with those of group C; daily sperm production in the testis of group H decreased markedly during week 3 of cadmium loading, and was significantly lower in groups M and H as compared to that in group C during week 6; alkaline phosphatase (ALP) in group H and lactate dehydrogenase-X (LDH-X) in groups M and H were markedly lower compared to those of group C; plasma testosterone (T) level in both cadmium loading groups decreased and was low or significantly lower than that in group C; follicle stimulating hormone (FSH) and luteinizing hormone (LH) levels had no apparent difference between the three groups. It is suggested that the gradual accumulation of cadmium in testis tissue induced by chronic cadmium loading results in changes in some enzyme activity, a decrease in sperm production, and defect of endocrine function activity in the testis.