Investigations on the Transfer of Quinolizidine Alkaloids from Lupinus angustifolius into the Milk of Dairy Cows.

Lupin varieties with a low content of quinolizidine alkaloids (QAs) like blue sweet lupin (BSL) have long been used as a protein source for dairy cows. A health concern for humans may arise from the transfer of acute toxic QAs from feed into cow's milk. This study is the first to quantify the transfer of QAs from BSL into cow's milk with experimental and modeling methods. Four lactating dairy cows were subjected to two 7 day feeding periods with 1 and 2 kg/d BSL, respectively, each followed by a depuration period. BSL contained 1774 mg/kg dry matter total QAs. Individual milk samples were taken twice daily and QA contents in feed and milk determined with liquid chromatography-tandem mass spectrometry. Transfer of QAs into the milk was already seen with the administration of 1 kg/d BSL, with differences in transfer rates (TRs) between individual QAs. A toxicokinetic model was derived to quantify and predict QA feed-to-food transfer. For the four most prominent QAs, our model shows an α-half-life of around 0.27 d. TRs were obtained for six QAs and were between 0.13 (sparteine) and 3.74% (multiflorine). A toxicological assessment of milk containing QAs as measured in this study indicated a potential health concern.

Biosynthesis of quinolizidine alkaloids in lupins: mechanistic considerations and prospects for pathway elucidation.

Covering: up to 2022Quinolizidine alkaloids (QAs) are a class of alkaloids that accumulate in a variety of leguminous plants and have applications in the agricultural, pharmaceutical and chemical industries. QAs are notoriously present in cultivated lupins (Lupinus spp.) where they complicate the use of the valuable, high-protein beans due to their toxic properties and bitter taste. Compared to many other alkaloid classes, the biosynthesis of QAs is poorly understood, with only the two first pathway enzymes having been discovered so far. In this article, we review the different biosynthetic hypotheses that have been put forth in the literature (1988-2009) and highlight one particular hypothesis (1988) that agrees with the often ignored precursor feeding studies (1964-1994). Our focus is on the biosynthesis of the simple tetracyclic QA (-)-sparteine, from which many of the QAs found in lupins derive. We examine every pathway step on the way to (-)-sparteine and discuss plausible mechanisms, altogether proposing the involvement of 6-9 enzymes. Together with the new resources for gene discovery developed for lupins in the past few years, this review will contribute to the full elucidation of the QA pathway, including the identification and characterization of the missing pathway enzymes.

Resolution of alkaloid racemate: a novel microbial approach for the production of enantiopure lupanine via industrial wastewater valorization.

BACKGROUND: Lupanine is a plant toxin contained in the wastewater of lupine bean processing industries, which could be used for semi-synthesis of various novel high added-value compounds. This paper introduces an environmental friendly process for microbial production of enantiopure lupanine. RESULTS: Previously isolated P. putida LPK411, R. rhodochrous LPK211 and Rhodococcus sp. LPK311, holding the capacity to utilize lupanine as single carbon source, were employed as biocatalysts for resolution of racemic lupanine. All strains achieved high enantiomeric excess (ee) of L-(-)-lupanine (> 95%), while with the use of LPK411 53% of the initial racemate content was not removed. LPK411 fed with lupanine enantiomers as single substrates achieved 92% of D-(+)-lupanine biodegradation, whereas L-(-)-lupanine was not metabolized. Monitoring the transcriptional kinetics of the luh gene in cultures supplemented with the racemate as well as each of the enantiomers supported the enantioselectivity of LPK411 for D-(+)-lupanine biotransformation, while (trans)-6-oxooctahydro-1H-quinolizine-3-carboxylic acid was detected as final biodegradation product from D-(+)-lupanine use. Ecotoxicological assessment demonstrated that lupanine enantiomers were less toxic to A. fischeri compared to the racemate exhibiting synergistic interaction. CONCLUSIONS: The biological chiral separation process of lupanine presented here constitutes an eco-friendly and low-cost alternative to widely used chemical methods for chiral separation.

Bioconversion of alkaloids to high-value chemicals: Comparative analysis of newly isolated lupanine degrading strains.

This work explores the potential for development of a lupanine valorization process evaluating different isolated microorganisms for their capacity to metabolize the alkaloid. Ecotoxicological assessment demonstrated that lupanine is toxic for Vibrio fischeri and Daphnia magna exhibiting EC50 values of 89 mg L-1 and 47 mg L-1 respectively, while acting both as growth inhibitor for a monocotyledonous and as promoter for a dicotyledonous plant. Among the eight aerobic and anaerobic strains isolated and identified Rhodococcus rhodochrous LPK211 achieved 81% removal for 1.5 g L-1 lupanine, while no end-products were detected by NMR constituting a promising microorganism for lupanine biodegradation. Moreover, Rhodococcus ruber LPK111 and Rhodococcus sp. LPK311 exhibited 66% and 71% of removal respectively, including potential formation of lupanine N-oxide. Pseudomonas putida LPK411 reached 80% of lupanine removal and generated three fermentation products potentially comprising 17-oxolupanine and lupanine derivatives with open ring structures enabling the development of alkaloid valorization processes.

Can the genotype or phenotype of two polymorphic drug metabolising cytochrome P450-enzymes identify oral lichenoid drug eruptions?

BACKGROUND: Lichenoid drug eruptions (LDE) in the oral cavity are adverse drug reactions (ADR) that are impossible to differentiate from oral lichen planus (OLP) as no phenotypic criteria exist. Impaired function of polymorphic cytochrome 450-enzymes (CYPs) may cause increased plasma concentration of some drugs resulting in ADR/LDE. In an earlier study we did not find more patients with OLP (OLPs) with impaired CYP-genotype. OBJECTIVES: To test if more OLPs have an impaired CYP-phenotype than to be expected from the CYP-genotype and to find clinical criteria characterising oral LDE. METHODS: One hundred and twenty OLPs were genotyped for the most common polymorphisms of CYP2D6 and CYP2C19 that result in impaired function. One hundred and ten did a phenotype test of both enzymes. The exposure to drugs and polypharmacy and the CYP metabolism of the drugs were evaluated. The OLP manifestations were registered. RESULTS: The only difference in OLP manifestations was that patients with a CYP2D6 genotype with less than two fully functional alleles presented more asymmetrical OLP distribution in particular in non-medicated patients (P < 0.05). No more OLPs than expected from the genotype had a phenotype with reduced function. However, the established phenotypic categories could not differentiate between the genotypes with two or one fully functional allele. Nevertheless, among the patients with a phenotype with normal function the patients with only one functional allele had a statistically significant higher metabolic ratio compared to patients with two fully functional alleles (P < 0.05). CONCLUSION: It was not possible to identify LDE by impaired function of polymorphic CYPs.

[Phenotypes of oxidation and acetylation in Alzheimer's disease--preliminary report]. / Fenotyp oksydacji i fenotyp acetylacji w chorobie Alzheimera--doniesienie wstepne.

AIM: The relationship between genetically determined polymorphic oxidation and acetylation and susceptibility to some disease has aroused much interest. The aim of our study was to evaluate whether patients with Alzheimer's disease differ from healthy persons in their ability to oxidize sparteine and acetylate sulphadimidine as model substance. METHOD: Oxidation and acetylation phenotype were estimated in 20 patients with Alzheimer's disease. The control group consisted of 160 healthy volunteers for comparison of oxidation phenotype and 45 healthy subjects for comparison of acetylation phenotype. RESULTS: The phenotyping of oxidation revealed two distinct populations among 20 patients with Alzheimer's disease: 19 persons (95%) were extensive metabolizers (EMs) of sparteine and 1 person (5%) was a poor metabolizer (PMs). In 160 healthy persons, 146 persons (91.2%) were extensive metabolizers of sparteine and 14 persons (8.8%) were poor metabolizers. The difference between the frequency distribution of PMs and EMs in healthy persons and in patients with Alzheimer's disease was not statistically significant. The phenotyping of acetylation showed that among 20 patients with Alzheimer's disease 10 persons (50%) were rapid acetylators and 10 persons (50%) were slow acetylators. In 45 healthy subjects the phenotype of rapid acetylation was observed in 23 persons (5 1%) and slow acetylation in 22 persons (49%). Our study showed a lack of statistically significant differences between the percentage of rapid acetylators (51%) and of slow acetylators (49%) in the control group of healthy volunteers and in the group ofAlzheimer's disease. CONCLUSION: The results of our study may suggest that phenotypes of oxidation and acetylation are not associated with risk of the development of Alzheimer's disease.

A natural variant of the heme-binding signature (R441C) resulting in complete loss of function of CYP2D6.

A new variant allele CYP2D6*62 (g.4044C>T; R441C) of the drug-metabolizing cytochrome P450 (P450) CYP2D6 was identified in a person with reduced sparteine oxidation phenotype, which was unexpected based on a genetic CYP2D6*1A/*41 background. The recombinantly expressed variant protein had no activity toward propafenone as a result of missing heme incorporation. Sequence alignment revealed that the positively charged R441 residue is part of the heme-binding signature but not strictly conserved among all the P450s. A compilation of described P450 monooxygenase variants revealed that other enzymes can functionally tolerate even nonconservative amino acid changes at the corresponding position (i.e., the invariant cysteine 2). This suggests that heme binding in mammalian P450s depends not only on the integrity of the heme-binding signature but also on other family- and subfamily-specific sequence determinants.

[Clinical significance of oxidation and acetylation genetic polymorphism in patients with hyperthyreosis]. / Kliniczne znaczenie fenotypu oksydacji i fenotypu acetylacji u chorych na nadczynnosc tarczycy.

INTRODUCTION: The relationship between genetically determined polymorphic oxidation and acetylation and susceptibility to some disease was aroused much interest. The aim of our study was to evaluate whether patients with hyperthyreosis differ from healthy persons in their ability to oxidize sparteine and acetylate sulphadimidine as model drugs. Oxidation and acetylation were estimated in 48 patients with hiperthyreosis. MATERIAL AND METHODS: The control group consisted of 160 healthy volunteers for comparison of oxidation phenotype and 60 healthy volunteers for comparison of acetylation phenotype. The phenotyping of oxidation revealed two distinct populations among 40 patients with hyperthyreosis: 38 persons (95%) were extensive metabolizers (EM) of sparteine and 2 persons (5%) was poor metabolizers (PM). In 160 healthy persons (91.2%) were EM and 14 persons (8.8%) were PM. The difference between frequency distribution of PM and EM in healthy persons and in patients with hyperthyreosis was not statistically significant. RESULTS: The phenotyping of acetylation showed among 48 patients with hyperthyreosis 8 persons (13%) were rapid acetylators (RA) and 40 persons (87%) were slow acetylators (SA). In 60 healthy volunteers the phenotype of rapid acetylation was observed in 31 persons (51%) and slow acetylation in 29 persons (49%). Relative risk (odds ratio) of development of thyroid diseases was 5.34 times higher for SA in comparison to RA. The prevalence of SA among patients with hyperthyreosis in comparison to healthy volunteers was statistically significant (p < 0.0002). CONCLUSIONS: The results of our study may suggest that slow acetylation phenotype is associated with increased risk of the development of hyperthyreosis.

Acute hypoxia and cytochrome P450-mediated hepatic drug metabolism in humans.

OBJECTIVE: Our objective was to investigate the effect of acute hypoxia on the activity of hepatic cytochrome P450 (CYP) enzymes. METHODS: Twelve healthy subjects who lived at sea level were exposed to altitude-induced hypoxia for 7 days at 4559 m above sea level. Hepatic CYP enzyme activity was measured before departure, at 24 and 96 hours after arrival to high-altitude location, and at 1 month after return to sea level. CYP enzyme activities were measured by means of the metabolic ratios of sparteine (CYP2D6), endogenous cortisol metabolism (CYP3A4), and caffeine (CYP1A2), as well as by the S/R ratio of mephenytoin (CYP2C19) and antipyrine clearance. RESULTS: The metabolic ratio of sparteine increased after 24 hours at high altitude (median difference, 0.15; 95% confidence interval, 0.05 to 0.28) and remained increased after 96 hours. The ratio decreased after return to sea level (median difference, -0.15; 95% confidence interval, -0.29 to -0.03; P =.016, Friedman test). The metabolic ratio of cortisol decreased after 24 hours (median difference, -2.0; 95% confidence interval, -3.5 to -0.5) but returned to sea level values after 96 hours at high altitude (median difference, 1.6; 95% confidence interval, 1.0 to 4.2; P =.047, Friedman test). These changes indicate a small decrease in the activity of CYP2D6 and CYP3A4. There were no significant changes regarding the metabolic ratio of caffeine, the S/R ratio of mephenytoin, or antipyrine clearance. CONCLUSION: The small changes observed suggest that acute hypoxia has no clinically significant effects on CYP enzymes in humans.

CYP2D6 extensive, intermediate, and poor phenotypes and genotypes in a Polish population.

OBJECTIVE: This study investigated the distribution of the CYP2D6 genotypes and phenotypes in a Polish population and compared the concordance of the two methods. METHODS: Six hundred unrelated healthy individuals from southwestern Poland were studied. The CYP2D6 phenotype was analyzed in 300 individuals using sparteine as a model drug. The CYP2D6 genotype was analyzed in 300 individuals by polymerase chain reaction amplification and restriction fragment length polymorphism techniques for the CYP2D6*1, CYP2D6*3, and CYP2D6*4 alleles. Additionally, in 60 randomly selected healthy individuals both the CYP2D6 phenotype and genotype was assessed to determine accordance between the methods. RESULTS: Of 300 participants in the study 25 (8.3%) were classified as poor metabolizers, 44 (14.7%) as intermediate metabolizers, and 231 (77%) as extensive metabolizers of sparteine. The frequency of CYP2D6*1, CYP2D6*3, and CYP2D6*4 alleles among the genotyped 300 persons was 75.7%, 1.3%, and 23.0%, respectively. The frequency of CYP2D6 deficient genotypes in a Polish population (8.0%) was similar to phenotyping results. The comparison of phenotype and genotype in 60 randomly selected individuals showed a good concordance of the obtained results. CONCLUSIONS. The frequencies of poor metabolizers for CYP2D6 phenotype (8.3%) and genotype (8.0%) in a Polish population from the southwestern region are in concordance and compare well with most results of poor oxidation metabolizers in other white populations.

Influence of CYP2D6 genotype and medication on the sparteine metabolic ratio of psychiatric patients.

OBJECTIVE: To investigate the influence of CYP2D6 genotype and medication on the reliability of phenotyping in a naturalistic setting of psychiatric inpatients. METHODS: The phenotype of 160 psychiatric inpatients was estimated by taking the urinary metabolic ratio (MR) of the concentrations of sparteine to 2- and 5-dehydrosparteine. Genotyping identified CYP2D6*1, *3, *4, *5 and *6 alleles as well as duplication of the CYP2D6 gene. All subjects underwent detailed drug history including drug dose and therapeutic drug monitoring to control compliance and abuse of other psychotropic drugs. These data were compared with those of 195 unmedicated healthy Germans. RESULTS: The cumulative distribution of the MR in patients showed a significant shift to higher MR when compared with that of healthy subjects (P < or = 0.001). Patients medicated either with selective serotonin reuptake inhibitors (SSRIs, P < or = 0.001), antipsychotic drugs (P= 0.002) or other drugs known to be substrates or inhibitors of CYP2D6 (P < or = 0.001) showed a significantly higher mean MR than unmedicated patients. However, there was no significant effect of tricyclic antidepressants on the MR. Healthy subjects with CYP2D6 deficiency were separated by a MR of greater than 20 from those who expressed functional CYP2D6. Seven patients carrying at least one functional CYP2D6 allele revealed a MR of greater than 20, indicating the occurrence of phenocopying. CONCLUSION: The results of phenotyping may be falsified by drugs known to be substrates or inhibitors of CYP2D6; thus, this method is not sufficiently reliable. However, since we observed the phenomenon of phenocopying only in patients treated with a SSRI such as fluoxetine, fluvoxamine or paroxetine, we conclude that sparteine phenotyping of medicated patients detects CYP2D6 deficiency correctly, provided that patients treated with these SSRIs are excluded.

Duplication of CYP2D6 predicts high clearance of desipramine but high clearance does not predict duplication of CYP2D6.

OBJECTIVE: Duplication of CYP2D6 causes very rapid metabolism of CYP2D6 substrates such as desipramine. However, we have previously shown that in the Danish population, only about 15% of very rapid metabolisers, defined as subjects with a metabolic ratio of sparteine of 0.15 or less, carried a duplicated allele. The question is whether gene duplication is a relatively rare cause (perhaps predictor) of very rapid metabolism or whether a low metabolic ratio is a poor predictor of this. METHODS: After measuring metabolic ratios anew, we selected six volunteers with duplication of CYP2D6 and metabolic ratios ranging from 0.07 to 0.17 and six volunteers without duplication with metabolic ratios ranging from 0.08 to 0.21. Each subject took 100 mg of desipramine. Blood and urine were collected for 48 h. RESULTS: The median total oral clearance of desipramine was 372 l/h and 196 l/h [median difference 108 l/h (95.9% c.i., -304-598 l/h)] and the median partial clearance of desipramine by 2-hydroxylation was 155 l/h and 87 l/h [median difference 47 l/h (95.9% c.i., -124-141 l/h)] for the group with duplication and the group without duplication, respectively. CONCLUSION: The predictive value of duplication of CYP2D6 is poor; there must be other causes (or predictors) of very rapid metabolism and with much higher frequency than duplication of CYP2D6.

Novel sparteine-mediated enantio-dichotomic formal synthesis of R-(-)- and S-(+)-curcuphenol.

High and opposite enantiodiscriminations were observed between tertiary amides and secondary amides in the sparteine-mediated lateral metalation-allylation of 2-ethyl-m-toluamide derivatives (2a, 2e). The results described above have been applied for the formal synthesis of both enantiomers of curcuphenol. The brief mechanistic studies suggested that stereoinformation was introduced after the deprotonation step.

Mass spectrometry of bis-quinolizidine alkaloids: 2- and 17-alkyl-substituted derivatives of sparteine and lupanine.

The mass spectral fragmentations of 2-methylsparteine (1), 2, 17-dimethylsparteine (2), 2-methyl-17-isopropylsparteine (3), 2-methyl-17-oxosparteine (4), 2-oxo-17-methylsparteine (17-methyllupanine) (5) and 2-oxo-17-isopropylsparteine (17-isopropyllupanine) (6) were investigated. Fragmentation pathways, whose identification was assisted by accurate mass measurements and a correlation between the abundances of the M(+.) and selected fragment ions of the investigated compounds, are discussed. The data obtained create the basis for distinguishing the structural isomers and metamers.

[Oxidation phenotype as a risk factor for development of allergic diseases]. / Fenotyp oksydacji jako czynnik zwiekszajacy ryzyko wystapienia chorób alergicznych.

The relationship between genetically determined polymorphic metabolism and susceptibility to allergic diseases has aroused much interest. The aim of our study was to evaluate whether patients with allergic diseases, like atopic asthma and allergic rhinitis differ from healthy persons in their ability to oxidize sparteine as a model drug. The study was completed by 200 persons, 40 patients with allergic diseases--20 with atopic asthma and 20 with allergic rhinitis and 160 healthy volunteers as a control group. The results of our study revealed a predominance of very extensive metabolizers of sparteine among patients with allergic diseases in comparison with healthy volunteers. The difference in the oxidation metabolic ratio (MR) frequency distribution between patients with allergic diseases and healthy persons was statistically significant. Relative risk (odds ratio) of development of atopic asthma was 3.29 times higher, and that of allergic rhinitis 2.94 times higher for persons with very extensive oxidation phenotype. Our results represent some evidence for a possible relationship between extensive, rapid oxidation phenotype and the higher susceptibility to development of atopic asthma and allergic rhinitis.

Phenotypes and genotypes for CYP2D6 and CYP2C19 in a black Tanzanian population.

AIMS: CYP2D6 and CYP2C19 are polymorphically expressed enzymes that show marked interindividual and interethnic variation. The aim of this study was to determine the frequency of the defective alleles in CYP2D6 and CYP2C19 in Africans and to test whether the genotype for CYP2C19 is better correlated with the proguanil/cylcoguanil ratio than the mephenytoin S/R ratio. METHODS: Two hundred and sixteen black Tanzanians were phenotyped for CYP2D6 with the use of sparteine, and for CYP2C19 with the use of mephenytoin and proguanil. Of these 196 subjects were also genotyped for CYP2D6 (including the CYP2D6*1, CYP2D6*3 and CYP2D6*4 alleles) and 195 were genotyped for CYP2C19 (including the CYP2C19*1, CYP2C19*2 and the CYP2C19*3 alleles). Furthermore 100 subjects were examined for the allele duplication in CYP2D6, leading to ultrarapid metabolism, with long PCR. RESULTS: The sparteine metabolic ratio (MR) was statistically significantly higher in the Tanzanian group of homozygous, extensive metabolizers compared to a historical control group of white Danish extensive metabolizers. Only one poor metabolizer for CYP2D6 (MR=124 and genotype CYP2D6*1/CYP2D6*4 ) was found. The gene frequencies were 0.96 for the CYP2D6*1 allele and 0.04 for the CYP2D6*4 allele. No CYP2D6*3 alleles were found. Nine subjects had an allele duplication in CYP2D6 (9%). For CYP2C19 there were seven subjects (3. 6%) who were phenotyped as poor metabolizers, but only three subjects (1.5%) had a genotype (CYP2C19*2/CYP2C19*2 ) indicative of poor metabolism. The gene frequencies were 0.90 for the CYP2C19*1 allele and 0.10 for the CYP2C19*2 allele. No CYP2C19*3 alleles were found. The mephenytoin S/R ratios were not bimodally distributed. CONCLUSIONS: Both the genotyping and phenotyping results show that there is a substantial difference between an African black population and a Caucasian population in the capacity to metabolize drugs via CYP2D6 and CYP2C19.

Clomipramine dose-effect study in patients with depression: clinical end points and pharmacokinetics. Danish University Antidepressant Group (DUAG).

OBJECTIVE: To examine the problems of establishing dose-effect and concentration-effect relationships of antidepressant therapy with clomipramine. METHODS: This randomized double-blind study compared five fixed doses of clomipramine hydrochloride: 25, 50, 75, 125, and 200 mg/day in hospitalized or day patients at nine clinical centers in Denmark. A 1-week washout period was followed by 6 weeks of active treatment and weekly depression ratings. In total, 151 patients (100 women and 51 men) with major depression scoring > or =18 on the Hamilton Depression Scale (HDS) or > or =9 on the Hamilton Depression subscale (HDSS) before and after the washout period were randomized. The treatment groups (n = 29 to 32) were well balanced with respect to sex, age, and depression rating. Serum concentrations of clomipramine plus metabolites were measured at weekly intervals. A sparteine test was performed before and during drug treatment. RESULTS: There was pronounced interpatient variability in response and kinetics at each dose. Drop-outs attributable to adverse events increased with rising doses, whereas drop-outs caused by worsening or lack of effect or nonresponse declined with increasing dose. Completer analyses showed a moderate and statistically significant relationship between depression rating and dose at all ratings after 1 to 6 weeks of treatment (trend analysis). HDS items representing core symptoms of depression showed a particularly consistent dose-effect relationship. Early sustained response occurred more frequently with the two highest doses. Serum levels of clomipramine and desmethylclomipramine showed weak correlation with depression ratings (Rs = -0.18 to -0.27; P < .05 to P < .01). A few blood pressure measurements and a few typical side-effect ratings showed a statistically significant dose-effect and concentration-effect relationship. Serum concentration of clomipramine and desmethylclomipramine showed a pronounced disproportionate increase with increasing dose. Clomipramine inhibited in a dose-dependent fashion CYP2D6 (sparteine oxidation). CONCLUSION: The dose-effect curves, indicating the probability of a certain outcome at a given dose, were flat and overlapping suggesting a narrow therapeutic range. This pattern is similar to that observed with newer antidepressants.