Poly(3-hydroxybuyrate) production from industrial hemp waste pretreated with a chemical-free hydrothermal process.

In this study, a mild two-stage hydrothermal pretreatment was employed to optimally valorize industrial hemp (Cannabis sativa sp.) fibrous waste into sugars for Poly(3-hydroxybuyrate) (PHB) production using recombinant Escherichia coli LSBJ. Biomass was pretreated using hot water at 160, 180, and 200 °C for 5 and 10 min (15% solids), followed by disk refining. The sugar yields during enzymatic hydrolysis were found to improve with increasing temperature and the yields for hot water-disk refining pretreatment (HWDM) were higher compared to only hot water pretreatment at all conditions. The maximum glucose (56 g/L) and cellulose conversion (92%) were achieved for HWDM at 200 °C for 10 min. The hydrolysate obtained was fermented at a sugar concentration of 20 g/L. The PHB inclusion and concentration of 48% and 1.8 g/L, respectively, were similar to those from pure sugars. A pH-controlled fermentation resulted in a near bi-fold increase in PHB yield (3.46 g/L).

Engineered Sorghum Bagasse Enables a Sustainable Biorefinery with p-Hydroxybenzoic Acid-Based Deep Eutectic Solvent.

Integrating multidisciplinary research in plant genetic engineering and renewable deep eutectic solvents (DESs) can facilitate a sustainable and economic biorefinery. Herein, we leveraged a plant genetic engineering approach to specifically incorporate C6 C1 monomers into the lignin structure. By expressing the bacterial ubiC gene in sorghum, p-hydroxybenzoic acid (PB)-rich lignin was incorporated into the plant cell wall while this monomer was completely absent in the lignin of the wild-type (WT) biomass. A DES was synthesized with choline chloride (ChCl) and PB and applied to the pretreatment of the PB-rich mutant biomass for a sustainable biorefinery. The release of fermentable sugars was significantly enhanced (∼190 % increase) compared to untreated biomass by the DES pretreatment. In particular, the glucose released from the pretreated mutant biomass was up to 12 % higher than that from the pretreated WT biomass. Lignin was effectively removed from the biomass with the preservation of more than half of the ß-Ο-4 linkages without condensed aromatic structures. Hydrogenolysis of the fractionated lignin was conducted to demonstrate the potential of phenolic compound production. In addition, a simple hydrothermal treatment could selectively extract PB from the same engineered lignin, showing a possible circular biorefinery. These results suggest that the combination of PB-based DES and engineered PB-rich biomass is a promising strategy to achieve a sustainable closed-loop biorefinery.

Enhanced polyhydroxybutyrate production from acid whey through determination of process and metabolic limiting factors.

To sustainably produce biodegradable polyhydroxybutyrate (PHB), this study investigated effects of process and metabolic limiting factors during bioconversion of acid whey (AW) to PHB, offering economic and environmental advantages for dairy industry. Recombinant Escherichia coli LSBJ was used to achieve high PHB yields by utilizing both lactose and lactic acid as carbon source. Up to 85% PHB accumulation was achieved during growth on the synthetic AW. Growth on raw AW had the highest PHB yield of 4 g/L and a high substrate utilization efficiency (95%). Notably, ratios of lactate: lactose and C/N impacted metabolic flux and PHB yields. Maintaining the fermentation pH enhanced PHB production. Furthermore, additives of inorganic nitrogen sources, minerals and trace metals promoted PHB production from AW. The study improves the understanding of factors affecting utilization of AW and demonstrated the high PHB yields using recombinant E. coli that could be leveraged to design a sustainable process.

Biopolymer poly-hydroxyalkanoates (PHA) production from apple industrial waste residues: A review.

Apple pomace, the residue which is left out after processing of apple serves as a potential carbon source for the production of biopolymer, PHA (poly-hydroxyalkanoates). It is rich in carbohydrates, fibers and polyphenols. Utilization of these waste resources has dual societal benefit-waste management and conversion of waste to an eco-friendly biopolymer. This will lower the overall economics of the process. A major limitation for the commercialization of biopolymer in comparison with petroleum derived polymer is the high cost. This article gives an overview of valorization of apple pomace for the production of biopolymer, various strategies adopted, limitations as well as future perspectives.

Experimental investigation of the adsorption and desorption of cellulase enzymes on zeolite-ß for enzyme recycling applications.

The recyclability of cellulase enzymes using zeolite and polyethylene glycol (PEG) was investigated. The cellulase enzymes from cellulose hydrolysate suspensions were adsorbed onto zeolite-ß under typical working conditions (pH 5). PEG having a molecular weight of 200 Da and 20 kDa was used as an eluent to desorb the cellulase enzymes from zeolite-ß. Adsorption and desorption profiles of cellulase enzymes were studied by varying pH, PEG concentration, and salt concentration. Maximum binding capacity, qm of the zeolite decreased by increasing the pH, or by introducing PEG. At pH 5, the qm of the zeolite was determined to be 121 × 10-3 g/g. About 24%, 51% and 75% of the adsorbed enzyme can be recovered using 1 M NaCl, PEG 200 and PEG 20000, respectively. The specific activity of the recovered enzyme increased by 57% due to the presence of residual PEG.

Effect of P450 Oxidoreductase Polymorphisms on the Metabolic Activities of Ten Cytochrome P450s Varied by Polymorphic CYP Genotypes in Human Liver Microsomes.

Background/ Aims: Little is known about the effect of P450 oxidoreductase (POR) gene polymorphisms on the activities of CYPs with multiple genotypes. METHODS: We genotyped 102 human livers for 18 known POR single nucleotide polymorphisms (SNPs) with allelic frequencies greater than 1% as well as for 27 known SNPs in 10 CYPs. CYP enzyme activities in microsomes prepared from these livers were determined by measuring probe substrate metabolism by high performance liquid chromatograph. RESULTS: We found that the effects of the 18 POR SNPs on 10 CYP activities were CYP genotype-dependent. The POR mutations were significantly associated with decreased overall Km for CYP2B6 and 2E1, and specific genotypes within CYP1A2, 2A6, 2B6, 2C8, 2D6 and 2E1 were identified as being affected by these POR SNPs. Notably, the effect of a specific POR mutation on the activity of a CYP genotype could not be predicted from other CYP genotypes of even the same CYP. When combining one POR SNP with other POR SNPs, a hitherto unrecognized effect of multiple-site POR gene polymorphisms (MSGP) on CYP activity was uncovered, which was not necessarily consistent with the effect of either single POR SNP. CONCLUSIONS: The effects of POR SNPs on CYP activities were not only CYP-dependent, but more importantly, CYP genotype-dependent. Moreover, the effect of a POR SNP alone and in combination with other POR SNPs (MSGP) was not always consistent, nor predictable. Understanding the impact of POR gene polymorphisms on drug metabolism necessitates knowing the complete SNP complement of POR and the genotype of the relevant CYPs.

Prediction of cytochrome P450-mediated drug clearance in humans based on the measured activities of selected CYPs.

Determining drug-metabolizing enzyme activities on an individual basis is an important component of personalized medicine, and cytochrome P450 enzymes (CYPs) play a principal role in hepatic drug metabolism. Herein, a simple method for predicting the major CYP-mediated drug clearance in vitro and in vivo is presented. Ten CYP-mediated drug metabolic activities in human liver microsomes (HLMs) from 105 normal liver samples were determined. The descriptive models for predicting the activities of these CYPs in HLMs were developed solely on the basis of the measured activities of a smaller number of more readily assayed CYPs. The descriptive models then were combined with the Conventional Bias Corrected in vitro-in vivo extrapolation method to extrapolate drug clearance in vivo. The Vmax, Km, and CLint of six CYPs (CYP2A6, 2C8, 2D6, 2E1, and 3A4/5) could be predicted by measuring the activities of four CYPs (CYP1A2, 2B6, 2C9, and 2C19) in HLMs. Based on the predicted CLint, the values of CYP2A6-, 2C8-, 2D6-, 2E1-, and 3A4/5-mediated drug clearance in vivo were extrapolated and found that the values for all five drugs were close to the observed clearance in vivo The percentage of extrapolated values of clearance in vivo which fell within 2-fold of the observed clearance ranged from 75.2% to 98.1%. These findings suggest that measuring the activity of CYP1A2, 2B6, 2C9, and 2C19 allowed us to accurately predict CYP2A6-, 2C8-, 2D6-, 2E1-, and 3A4/5-mediated activities in vitro and in vivo and may possibly be helpful for the assessment of an individual's drug metabolic profile.

Gene polymorphisms and contents of cytochrome P450s have only limited effects on metabolic activities in human liver microsomes.

Extensive inter-individual variations in pharmacokinetics are considered as a major reason for unpredictable drug responses. As the most important drug metabolic enzymes, inter-individual variations of cytochrome P450 (CYP) activities are not clear in human liver. In this paper, metabolic activities, gene polymorphisms and protein contents of 10 CYPs were determined in 105 human normal liver microsomes. The results indicated substantial inter-individual variations in CYP activities, with the greatest being CYP2C19 activity (>600-fold). Only half of 10 CYP isoforms and 26 gene polymorphism sites had limited effects on metabolic activities, such as CYP2A6, CYP2B6, CYP2C9, CYP2D6 and CYP3A4/5, others had almost no effects. Compared with their respective wild type, Km, Vmax, and CLint decreased by 51.6%, 88.7% and 70.7% in CYP2A6*1/*4 genotype, Vmax and CLint decreased by 32.8% and 60.2% in CYP2C9*1/*3 genotype, Km increased by 118.4% and CLint decreased by 65.2% in CYP2D6 100TT genotype, respectively. Moreover, there were only 4 CYP isoforms, CYP1A2, CYP2A6, CYP2E1 and CYP3A5, which had moderate or weak correlations between Vmax values and corresponding contents. In conclusions, the genotypes and contents of some CYPs have only limited effects on metabolic activities, which imply that there are other more important factors to influence inter-individual variations.

Correlation of Cytochrome P450 Oxidoreductase Expression with the Expression of 10 Isoforms of Cytochrome P450 in Human Liver.

Human cytochrome P450 oxidoreductase (POR) provides electrons for all microsomal cytochromes P450 (P450s) and plays an indispensable role in drug metabolism catalyzed by this family of enzymes. We evaluated 100 human liver samples and found that POR protein content varied 12.8-fold, from 12.59 to 160.97 pmol/mg, with a median value of 67.99 pmol/mg; POR mRNA expression varied by 26.4-fold. POR activity was less variable with a median value of 56.05 nmol/min per milligram. Cigarette smoking and alcohol consumption clearly influenced POR activity. Liver samples with a 2286822 TT genotype had significantly higher POR mRNA expression than samples with CT genotype. Homozygous carriers of POR2286822C>T, 2286823G>A, and 3823884A>C had significantly lower POR protein levels compared with the corresponding heterozygous carriers. Liver samples from individuals homozygous at 286823G>A, 1135612A>G, and 10954732G>A generally had lower POR activity levels than those from heterozygous or wild-type samples, whereas the common variant POR*28 significantly increased POR activity. There was a strong association between POR and the expression of P450 isoforms at the mRNA and protein level, whereas the relationship at the activity level, as well as the effect of POR protein content on P450 activity, was less pronounced. POR transcription was strongly correlated with both hepatocyte nuclear factor 4 alpha and pregnane X receptor mRNA levels. In conclusion, we have elucidated some potentially important correlations between POR single-nucleotide polymorphisms and POR expression in the Chinese population and have developed a database that correlates POR expression with the expression and activity of 10 P450s important in drug metabolism.

Significant change of cytochrome P450s activities in patients with hepatocellular carcinoma.

The lack of information concerning individual variation in drug-metabolizing enzymes is one of the most important obstacles for designing personalized medicine approaches for hepatocellular carcinoma (HCC) patients. To assess cytochrome P450 (CYP) in the metabolism of endogenous and exogenous molecules in an HCC setting, the activity changes of 10 major CYPs in microsomes from 105 normal and 102 HCC liver tissue samples were investigated. We found that CYP activity values expressed as intrinsic clearance (CLint) differed between HCC patients and control subjects. HCC patient samples showed increased CLint for CYP2C9, CYP2D6, and CYP2E1 compared to controls. Meanwhile, CYP1A2, CYP2C8, and CYP2C19 CLint values decreased and CYP2A6, CYP2B6, and CYP3A4/5 activity was unchanged relative to controls. For patients with HCC accompanied by fibrosis or cirrhosis, the same activity changes were seen for the CYP isoforms, except for CYP2D6 which had higher values in HCC patients with cirrhosis. Moreover, CYP2D6*10 (100C>T), CYP2C9*3 (42614 A>C), and CYP3A5*3 (6986A>G) polymorphisms had definite effects on enzyme activities. In the HCC group, the CLint of CYP2D6*10 mutant homozygote was decreased by 95% compared to wild-type samples, and the frequency of this homozygote was 2.8-fold lower than the controls.In conclusion, the activities of CYP isoforms were differentially affected in HCC patients. Genetic polymorphisms of some CYP enzymes, especially CYP2D6*10, could affect enzyme activity. CYP2D6*10 allelic frequency was significantly different between HCC patients and control subjects. These findings may be useful for personalizing the clinical treatment of HCC patients as well as predicting the risk of hepatocarcinogenesis.

Changes in cytochrome P450s-mediated drug clearance in patients with hepatocellular carcinoma in vitro and in vivo: a bottom-up approach.

Hepatocellular carcinoma (HCC) accompanied by severe liver dysfunction is a serious disease, which results in altered hepatic clearance. Generally, maintenance doses depend upon drug clearance, so individual dosage regimens should be customized for HCC patients based on the condition of patients. Based on clearance of CYP isoform-specific substrates at the microsomal level (CLM), microsomal protein per gram of liver (MPPGL), liver weight, hepatic blood flow, hepatic clearance values (CLH) for 10 CYPs in HCC patients (n=102) were extrapolated using a predictive bottom-up pharmacokinetic model. Compared with controls, the CLM values for CYP2C9, 2D6, 2E1 were significantly increased in HCC patients. Additionally, CYP1A2, 2C8, 2C19 CLM values decreased while the values for CYP2A6, 2B6, 3A4/5 were unchanged. The MPPGL values in HCC tissues were significantly reduced. CLH values of HCC patients for CYP1A2, 2A6, 2B6, 2C8, 2C19, and 3A4/5 were significantly reduced, while this for CYP2E1 were markedly increased and those for CYP2C9 and 2D6 did not change. Moreover, disease (fibrosis and cirrhosis) and polymorphisms of the CYP genes have influenced the CLH for some CYPs. Prediction of the effects of HCC on drug clearance may be helpful for the design of clinical studies and the clinical management of drugs in HCC patients.

Content and activity of human liver microsomal protein and prediction of individual hepatic clearance in vivo.

The lack of information concerning individual variation in content and activity of human liver microsomal protein is one of the most important obstacles for designing personalized medicines. We demonstrated that the mean value of microsomal protein per gram of liver (MPPGL) was 39.46 mg/g in 128 human livers and up to 19-fold individual variations existed. Meanwhile, the metabolic activities of 10 cytochrome P450 (CYPs) were detected in microsomes and liver tissues, respectively, which showed huge individual variations (200-fold). Compared with microsomes, the activities of liver tissues were much suitable to express the individual variations of CYP activities. Furthermore, individual variations in the in vivo clearance of tolbutamide were successfully predicted with the individual parameter values. In conclusion, we offer the values for MPPGL contents in normal liver tissues and build a new method to assess the in vitro CYP activities. In addition, large individual variations exist in predicted hepatic clearance of tolbutamide. These findings provide important physiological parameters for physiologically-based pharmacokinetics models and thus, establish a solid foundation for future development of personalized medicines.

Effect of Cytochrome b5 Content on the Activity of Polymorphic CYP1A2, 2B6, and 2E1 in Human Liver Microsomes.

Human cytochrome b5 (Cyt b5) plays important roles in cytochrome P450 (CYP)-mediated drug metabolism. However, the expression level of Cyt b5 in normal human liver remains largely unknown. The effect of Cyt b5 on overall CYP activity in human liver microsomes (HLM) has rarely been reported and the relationship between Cyt b5 and the activity of polymorphic CYP has not been systematically investigated. In this study, we found that the median value of Cyt b5 protein was 270.01 pmol/mg from 123 HLM samples, and 12- and 19-fold individual variation was observed in Cyt b5 mRNA and protein levels, respectively. Gender and smoking clearly influenced Cyt b5 content. In addition, we found that Cyt b5 protein levels significantly correlated with the overall activity of CYP1A2, 2B6, and 2E1 in HLM. However, when the CYP activities were sorted by single nucleotide polymorphisms (SNP), the effect of Cyt b5 protein on the kinetic parameters varied greatly. There were significant correlations between Cyt b5 content and Vmax and CLint of CYP1A2 wild-types (3860GG, 2159GG, and 5347CC) as well as homozygous mutants (163AA and 3113GG). In contrast to Vmax and CLint, the Km of CYP2B6 516GG and 785AA genotypes was inversely associated with Cyt b5 content. Correlations between Cyt b5 content and Vmax and CLint of CYP2E1 -1293GG, -1293GC, 7632TT, 7632TA, -333TT, and -352AA genotypes were also observed. In conclusion, Cyt b5 expression levels varied considerably in the Chinese cohort from this study. Cyt b5 had significant impact on the overall activity of CYP1A2, 2B6, and 2E1 in HLM and the effects of Cyt b5 protein on polymorphic CYP1A2, 2B6, and 2E1 activity were SNP-dependent. These findings suggest that Cyt b5 plays an important role in CYP-mediated activities in HLM and may possibly be a contributing factor for the individual variation observed in CYP enzyme activities.

Inhibition of baicalin on metabolism of phenacetin, a probe of CYP1A2, in human liver microsomes and in rats.

Baicalin has been used as mainly bioactive constituent of about 100 kinds of traditional Chinese medicines in Chinese pharmacopoeia. The effect of baicalin on cytochrome P450 should be paid more attention because baicalin was used widely. The aim of this study was to investigate whether baicalin could inhibit CYP1A2 in pooled human liver microsomes (HLMs) and in rats in vivo and the gene polymorphisms could affect inter-individual variation in IC50 in 28 human livers. Phenacetin was used as probe of CYP1A2. Kinetic parameter of CYP1A2 and IC50 of baicalin on CYP1A2 to each sample were measured and the common CYP1A2 polymorphisms (-3860G>A and -163C>A) were genotyped. The results showed that baicalin exhibited a mixed-type inhibition in pooled HLMs, with a Ki value of 25.4 µM. There was substantial variation in Km, Vmax, CLint of CYP1A2 and IC50 of baicalin on CYP1A2 (3∼10-fold). The range was from 26.6 to 114.8 µM for Km, from 333 to 1330 pmol·min(-1)·mg(-1)protein for Vmax and from 3.8 to 45.3 µL·min(-1)·mg(-1) protein for CLint in HLMs (n = 28). The Mean (range) value of IC50 in 28 HLMs was 36.3 (18.9 to 56.1) µM. The genotypes of -3860G>A and -163C>A had no significant effect on the inhibition of baicalin on CYP1A2. The animal experiment results showed that baicalin (450 mg/kg, i.v.) significantly decreased the Cmax and CL of phenacetin, and increased C(60 min), t1/2, Vd and AUC (P<0.05). There were significant correlations between percentage of control in C(60 min), t1/2, CL, AUC of phenacetin and Cmax of baicalin in 11 rats (P<0.05). Protein binding experiments in vitro showed that baicalin (0-2000 mg/L) increased the unbound phenacetin from 14.5% to 28.3%. In conclusion, baicalin can inhibit the activity of CYP1A2 in HLMs and exhibit large inter-individual variation that has no relationship with gene polymorphism. Baicalin can change the pharmacokinetics of phenacetin in rats.

Contribution of baicalin on the plasma protein binding displacement and CYP3A activity inhibition to the pharmacokinetic changes of nifedipine in rats in vivo and in vitro.

Baicalin purified from the root of Radix scutellariae is widely used in clinical practices. This study aimed to evaluate the effect of baicalin on the pharmacokinetics of nifedipine, a CYP3A probe substrate, in rats in vivo and in vitro. In a randomised, three-period crossover study, significant changes in the pharmacokinetics of nifedipine (2 mg/kg) were observed after treatment with a low (0.225 g/kg) or high (0.45 g/kg) dose of baicalin in rats. In the low- and high-dose groups of baicalin-treated rats, C max of total nifedipine decreased by 40%±14% (P<0.01) and 65%±14% (P<0.01), AUC0-∞ decreased by 41%±8% (P<0.01) and 63%±7% (P<0.01), Vd increased by 85%±43% (P<0.01) and 224%±231% (P<0.01), and CL increased by 97%±78% (P<0.01) and 242%±135% (P<0.01), respectively. Plasma protein binding experiments in vivo showed that C max of unbound nifedipine significantly increased by 25%±19% (P<0.01) and 44%±29% (P<0.01), respectively, and there was a good correlation between the unbound nifedipine (%) and baicalin concentrations (P<0.01). Furthermore, in vitro results revealed that baicalin was a competitive displacer of nifedipine from plasma proteins. In vitro incubation experiments demonstrated that baicalin could also competitively inhibit CYP3A activity in rat liver microsomes in a concentration-dependent manner. In conclusion, the pharmacokinetic changes of nifedipine may be modulated by the inhibitory effects of baicalin on plasma protein binding and CYP3A-mediated metabolism.

Pharmacokinetic changes of unbound theophylline are due to plasma protein binding displacement and CYP1A2 activity inhibition by baicalin in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Baicalin is one of the major bioactive constituents of Scutellariae Radix, the root of Scutellariae baicalensis Georgi and possesses a wide variety of pharmacological properties. AIM OF THE STUDY: To elucidate the effect of baicalin on the pharmacokinetics of theophylline in rats, focusing on plasma protein binding displacement and inhibition effect on CYP1A2 in vivo and in vitro. MATERIALS AND METHODS: The study was a randomized, three-period crossover design. Nine rats were given saline (control) or 450 mg/kg baicalin (dosage regimen A or B). Dosage regimen A was administered once at 0 h. Dosage regimen B was divided into three dosages (225,112.5, 112.5 mg/kg) and was given at 0, 2 and 4 h, respectively. Then theophylline (5 mg/kg, i.v.) was administered immediately. The effect of baicalin on CYP1A2 activity was determined by metabolism of phenacetin in vitro and plasma protein binding of theophylline was determined by ultrafiltration. RESULTS: C(max) decreased from (12.4 ± 1.6) to (8.7 ± 0.9) and (8.6 ± 2.0) mg/L, T(1/2) increased by 116 and 96%, V(d) increased by 51 and 49% for total theophylline in rats treated with dosage regimen A and B of baicalin, respectively. Cmax was significantly increased, V(d) decreased by 43 and 29% for unbound theophylline in rats treated with dosage regimen A and B of baicalin, respectively (P < 0.01). T(1/2) of unbound theophylline increased by 104% only in rats treated with dosage regimen B. No significant effects on the CL and AUC of both total and unbound theophylline were observed in the rats treated with dosage regimen A, but the CL decreased and AUC increased for total theophylline and CL decreased for unbound theophylline in the group treated with dosage regimen B (P < 0.05). Correlation analysis showed that the mean unbound theophylline (%) and mean baicalin concentration was in good correlation (P < 0.01). Baicalin decreased metabolism of phenacetin and exhibited a mixed-type inhibition in rat liver microsomes, with a K(i) value of 88.1 µM in vitro. Moreover baicalin was a competitive displacer of theophylline from plasma protein in vitro. CONCLUSIONS: The changes in Cmax, T(1/2), CL and AUC of theophylline due to baicalin may be attributed to two mechanisms, plasma protein binding displacement and CYP1A2 activity inhibition.

Concentration-dependent inhibitory effects of baicalin on the metabolism of dextromethorphan, a dual probe of CYP2D and CYP3A, in rats.

Baicalin has been shown to possess many pharmacological effects, including antiviral, antioxidant, anti-cancer and anti-inflammatory properties. In the current study, we reveal the inhibitory effects of baicalin on the metabolism of dextromethorphan (DXM), a dual probe substrate of CYP2D and CYP3A, in rats. Lineweaver-Burk plots demonstrated that baicalin inhibited the activities of CYP2D and CYP3A in a non-competitive manner in rat liver microsomes (RLMs). Concomitant administration of baicalin (0.90 g/kg, i.v.) and DXM (10 mg/kg, i.v.) increased the maximum drug concentration (C(max)) (37%) and the area under concentration-time curve (AUC) (42%) and decreased the clearance (CL) (27%) of DXM in a randomised, crossover study in rats (P < 0.01). The change in the AUC of DXM was significantly correlated with the C(max) and AUC of baicalin (P < 0.05). The inhibitory effects of multiple doses of baicalin (0.90 g/kg, i.v., 12 days) on the metabolism of DXM were similar to those observed following a single dose in rats. The activity of CYP3A in excised liver samples from rats following multiple baicalin treatment was significantly decreased compared to that of the control group (P < 0.05), whereas multiple doses of baicalin had no obvious effect on the activity of CYP2D. Taken together, these data demonstrate that baicalin inhibits the metabolism of DXM in a concentration-dependent manner in rats, possibly through inhibiting hepatic CYP2D and CYP3A activities.

Polymorphisms and haplotype analysis of IL-4Ralpha Q576R and I75V in patients with penicillin allergy.

BACKGROUND: The interleukin (IL)-4 and IL-4R genes are linked to allergic diseases and atopy and elevated serum IgE levels closely follow the presentation of penicillin allergy. We have evaluated the association between specific immunoglobulin (Ig)E, polymorphisms of IL-4Ralpha, and penicillin allergy. METHODS: Eight types of specific IgE to penicillins were detected with the radioallergosorbent test (RAST) in 242 patients with penicillin allergy and 220 control subjects. Polymorphisms of IL-4Ralpha Q576R and IL-4Ralpha I75V were genotyped by PCR-restriction fragment length polymorphism (RFLP). The polymorphisms and haplotype of IL-4Ralpha Q576R and I75V were analyzed. RESULTS: For IL-4Ralpha Q576R, the frequency of the AA genotype was higher in patients with penicillin allergy (76 vs. 64%, P = 0.005). The A allele occurred more frequently in the penicillin-allergic patients than in the controls (87 vs. 80%, P = 0.003). For IL-4Ralpha I75V, the AA genotype was more likely to be detected in the urticaria group than in the control group (32 vs. 19%, P = 0.049). Haplotype analysis revealed that Q576/I75 was more frequently found in patients with penicillin allergy than in controls (42 vs. 35%, P = 0.037). CONCLUSIONS: The IL4Ralpha Q576 allele is related to penicillin allergy, and the IL-4Ralpha I75 allele is associated with the symptom of urticaria. The Q576/I75 haplotype may be related with an allergy to penicillin.

Specific IgG antibodies in sera in patients with penicillin allergy.

The role of IgG antibodies in inducing or modifying allergic reaction has not been sufficiently clarified. The objective of this investigation is to elucidate the relationship between IgG antibodies and penicillin allergy, between IgG and IgE antibodies in allergic patients. Enzyme-linked immunosorbent assay and Radioallergosorbent test were used to examine eight kinds of specific IgG and IgE antibodies, including major antigenic determinants: benzylpenicilloyl (BPO), ampicilloyl (APO), amoxicilloyl (AXO) and phenoxomethylpenicilloyl (PVO), and minor antigenic determinants: benzylpenicillanyl (BPA), ampicillanyl (APA), amoxicillanyl (AXA) and phenoxomethylpenicillany (PVA), in the sera of 249 patients with penicillin allergy. Except BPA-IgG, seven kinds of antigenic determinants IgG antibodies levels were significantly higher than that of control group (P < 0.05). Positive rates of specific IgG and IgE were 47.0 and 57.8%, while positive rate of IgE and IgG together was 77.9%. The positive rate of IgG antibodies to major antigenic determinants (42.2%) was significantly higher than that of minor antigenic determinants (8.8%) (P < 0.05). The positive rate of IgG antibodies of patients with typical clinical symptoms after penicillin administration when skin tests were negative was significantly higher than that of patients with positive skin test (P < 0.01). There were no differences between the IgG positive rates to three kinds of determinants and that of all of eight kinds. The study indicates that IgG may be important in penicillin allergy with negative skin test and IgG antibodies to major antigenic determinants probably play a more important role in the process of allergic reaction.

Relationships between specific serum IgE, IgG, IFN-gamma level and IFN-gamma, IFNR1 polymorphisms in patients with penicillin allergy.

OBJECTIVE: The findings of numerous studies have suggested that both genetic and environmental influences are involved in the pathogenesis of allergic disease and atopy. We studied the polymorphisms in the interferon (IFN)-gamma (gamma) and IFN-gamma receptor 1 (IFNR1) gene with the aim of clarifying the relationships among these polymorphisms, penicillin allergy and anti-penicillin antibodies. METHODS: A restriction endonuclease fragment length polymorphism (RFLP)-PCR analysis and sequencing were used to study the IFNR1 and IFN-gamma polymorphisms. The presence and level of eight specific immunoglobulin (Ig)E and IgG antibodies were determined by the radioallergosorbent test (RAST) and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: The positive rates of specific IgE and IgG were 61.11 and 53.92%, respectively. There was no significant difference in the whole-allele of IFN-gamma distribution between patients with a penicillin allergy and control subjects. Allele 7 (18CA repeat) was significantly less frequent in the urticaria group (3.19 vs. 11.93%) than in the controls. There was no difference in IFN-gamma production among different alleles in IFN-gamma. The frequency of G/A (Val/Met) in the IFNR1 gene in allergic patients was significantly less than that in the controls (P < 0.05). There were no significant differences in the positive rate of IgE among different alleles of IFN-gamma. The same was true for the positive rate of IgG. CONCLUSIONS: The Met/Val allele in IFNR1 gene may have a protective role in the non-penicillin allergic population. The allele 18CA repeat in IFN-gamma gene may be associated with urticaria.