Growth independent rhamnolipid production from glucose using the non-pathogenic Pseudomonas putida KT2440.

BACKGROUND: Rhamnolipids are potent biosurfactants with high potential for industrial applications. However, rhamnolipids are currently produced with the opportunistic pathogen Pseudomonas aeruginosa during growth on hydrophobic substrates such as plant oils. The heterologous production of rhamnolipids entails two essential advantages: Disconnecting the rhamnolipid biosynthesis from the complex quorum sensing regulation and the opportunity of avoiding pathogenic production strains, in particular P. aeruginosa. In addition, separation of rhamnolipids from fatty acids is difficult and hence costly. RESULTS: Here, the metabolic engineering of a rhamnolipid producing Pseudomonas putida KT2440, a strain certified as safety strain using glucose as carbon source to avoid cumbersome product purification, is reported. Notably, P. putida KT2440 features almost no changes in growth rate and lag-phase in the presence of high concentrations of rhamnolipids (> 90 g/L) in contrast to the industrially important bacteria Bacillus subtilis, Corynebacterium glutamicum, and Escherichia coli. P. putida KT2440 expressing the rhlAB-genes from P. aeruginosa PAO1 produces mono-rhamnolipids of P. aeruginosa PAO1 type (mainly C(10):C(10)). The metabolic network was optimized in silico for rhamnolipid synthesis from glucose. In addition, a first genetic optimization, the removal of polyhydroxyalkanoate formation as competing pathway, was implemented. The final strain had production rates in the range of P. aeruginosa PAO1 at yields of about 0.15 g/g(glucose) corresponding to 32% of the theoretical optimum. What's more, rhamnolipid production was independent from biomass formation, a trait that can be exploited for high rhamnolipid production without high biomass formation. CONCLUSIONS: A functional alternative to the pathogenic rhamnolipid producer P. aeruginosa was constructed and characterized. P. putida KT24C1 pVLT31_rhlAB featured the highest yield and titer reported from heterologous rhamnolipid producers with glucose as carbon source. Notably, rhamnolipid production was uncoupled from biomass formation, which allows optimal distribution of resources towards rhamnolipid synthesis. The results are discussed in the context of rational strain engineering by using the concepts of synthetic biology like chassis cells and orthogonality, thereby avoiding the complex regulatory programs of rhamnolipid production existing in the natural producer P. aeruginosa.

Atypical serum transferrin isoform distribution in liver cirrhosis studied by HPLC, capillary electrophoresis and transferrin genotyping.

BACKGROUND: An incomplete separation of disialotransferrin (CDT) and trisialotransferrin (a non-CDT isoform) may cause false-positive CDT results in alcohol abuse testing. We describe a currently unknown disialotransferrin-trisialotransferrin-bridging phenomenon (di-tri-bridge) appearing with high prevalence in serum from liver cirrhosis patients. METHODS: Twenty one consecutive serum samples with a di-tri-bridge encountered in routine CDT HPLC (Clin-Rep(R)-CDT-on-line, Recipe) were investigated by a candidate reference CDT HPLC method, by capillary electrophoresis (Capillarys-CDT, Sebia) and by transferrin genotyping. Patients clinical background was assessed by telephone interview. RESULTS: Out of 21 consecutive serum samples showing a di-tri-bridge (and increased trisialotransferrin fractions) in HPLC as well as in CE analysis, 19 were from patients with a liver cirrhosis history. Genotyping (where applicable by the availability of DNA: n=12) yielded most frequently homozygous transferrin C1 (6x), proving that the di-tri-bridge cannot be explained by genetic transferrin variants in these samples. Other genotypes found were C2 (1x), C1C2 (4x), C1C3 (1x). CONCLUSION: The frequently seen di-tri-bridging phenomenon in transferrin HPLC analysis for patients with liver cirrhosis is not explained by genetic transferrin variants or by an increased trisialotransferrin fraction. Although further studies are needed to assess the relationship between this phenomenon and liver cirrhosis, our observation could be helpful in development of a biomarker for liver cirrhosis.

High prevalence of increased trisialotransferrin concentrations in patients with anorexia nervosa: implications for determination of carbohydrate-deficient transferrin.

BACKGROUND: Carbohydrate-deficient transferrin (asialo-+monosialo-+disialotransferrin, CDT) is currently the most specific laboratory marker of chronic alcohol abuse. We tested whether previous findings of false-positive CDT results for anorexia nervosa patients have been due to invalid CDT analysis methods or anorexia nervosa by itself. METHODS: Serum CDT from 49 anorexia nervosa patients, 14 bulimia nervosa patients and 22 healthy controls (all adolescent, female and age-matched) was determined in a retrospective study by HPLC (Clin-Rep-CDT-in-serum-online, cut-off > or =1.8%, Recipe), by capillary electrophoresis (Capillarys-CDT, cut-off > or =1.3%, Sebia) and (due to limited surplus serum volume for a subset of 18 anorexia nervosa patients with increased trisialotransferrin detected by HPLC) by immunoassay based on anion-exchange CDT and non-CDT fractionation (%CDT-TIA, cut-off > or =2.6% CDT, Bio-Rad). RESULTS: HPLC and capillary electrophoresis: No false-positive CDT results were obtained. Asialo- and monosialotransferrin were not detected and disialotransferrin (CDT) was in each case clearly below the test-specific cut-offs. Trisialotransferrin (a non-CDT isoform) was increased (cut-off > or =5.0% for HPLC) in 33 anorexia patients, 2 bulimia patients and 2 controls. %CDT-TIA: 8 false-positive CDT results of > or =2.6% out of the 18 samples tested (CDT-range/mean/median 2.6-4.6/3.2/2.8%). CONCLUSIONS: Anorexia nervosa does not cause by itself increased CDT results. False-positive CDT values from the past are most likely due to an incomplete separation of trisialotransferrin from CDT and thus overdetermination of CDT. Immunological CDT testing without confirmatory analysis by HPLC or CE is no longer acceptable.

Paediatric age-dependent serum transferrin isoform distribution studied by HPLC.

BACKGROUND: Human serum transferrin shows different transferrin isoforms with e.g. a varying amount of glycosylation, resulting in asialo-, mono-, di-, up to octasialotransferrin. We wanted to examine whether there are age-dependent differences in this transferrin isoform distribution. METHODS: Serum samples from a total of 126 paediatric patients (mean/median/minimum/maximum: 6.8/6.0/0.5/14 years) grouped in seven age groups (<2 years, 3-4 years, up to 13-14 years) were analyzed on an HPLC (Recipe Chemicals & Instruments GmbH, Munich, Germany). Means, medians and percentiles were computed for each transferrin isoform and tested for statistically significant differences between the age groups. RESULTS: CDT corresponded to disialotransferrin (since asialo- and monosialotransferrins were not detectable) and did not show statistically significant differences between the 7 age groups. The latter is also true for trisialo- and tetrasialotransferrin whereas pentasialotransferrin shows a statistically significant decrease with age. CONCLUSIONS: We suggest that age-independent decision limits, e.g. the 95% percentiles for disialotransferrin (1.1%) and trisialotransferrin (5.3%), can be used for the differentiation between normal and increased fractions of these isoforms until paediatric reference ranges have been established. The presence of asialo- and monosialotransferrin in paediatric serum should be considered as abnormal.

Lessons learned from a case of tert-butyl glucuronide excretion in urine - "New" psychoactive alcohols knocking on the back door?

BACKGROUND: Ethyl glucuronide (EtG) in urine is considered a marker of recent ethanol consumption or ethanol exposition. tert-Butanol is primarily used as a solvent and intermediate chemical. Like tert-amyl alcohol, tert-butanol is discussed in Internet forums as ethanol replacement. We discuss false-positive immunological EtG screenings by excretion of different alcohol glucuronides (EtG homologs), mainly tert-butyl glucuronide in urine of a polytoxikomanic in-patient. METHODS: Three consecutive urine samples from an in-patient with a long history of multiple substance abuse including solvents were analyzed by DRI EtG enzyme immunoassay (ThermoFisher Scientific Microgenics) on a Beckman Coulter AU680 analyzer, an in-house LC-MS/MS for EtG, 1-propyl, 2-propyl, 1-butyl, 2-butyl, and tert-butyl glucuronide, and an in-house headspace GC-FID of free congener substances methanol, 1-propanol, 2-butanone, 2-butanol, isobutanol, 1-butanol, 3-methyl-1-butanol, 2-methyl-1-butanol, and additionally for ethanol, acetone, 2-propanol, tert-butanol and 2-methyl-2-butanol. RESULTS: EtG immunoassay yielded two positive urine samples (0.2 and 0.6mg/L or 0.1 and 0.2mg/g creatinine; cut-off 0.1mg/L) which were tested EtG negative by LC-MS/MS (cut-off 0.1mg/L) but positive for tert-butyl glucuronide (3.7 and 27.1mg/L), 2-butyl glucuronide (1.1 and 3.5mg/L), and 2-propyl glucuronide (0.1 and 0.4mg/L). Headspace GC-FID detected tert-butanol (0.97 and 4.01mg/L), methanol (0.96 and 0.62mg/L), 2-butanone (0.84 and 1.65mg/L), and 2-butanol (0.04 and 0.09mg/L), but no ethanol and no 2-methyl-2-butanol. CONCLUSION: Cross-reaction of EtG homologs, mainly tert-butyl glucuronide after suspected tert-butanol or isobutane abuse, explains the false-positive EtG immunoassay findings. Future investigations could address the usefulness of alcohol glucuronides (EtG homologs) in urine as (a) biomarkers of an exposition to alkans or their corresponding alcohol metabolites and (b) as markers for using "old"-well known alcohols like tert-butanol or tert-amyl alcohol as easy to obtain, cheap, potent and "undetectable" ethanol replacements or "New" Psychoactive Alcohols.

Reprint of Standardisation and use of the alcohol biomarker carbohydrate-deficient transferrin (CDT).

Carbohydrate-deficient transferrin (CDT) is a glycoform profile of serum transferrin that increases in response to sustained high alcohol intake and over the last decades has become an important alcohol biomarker with clinical and forensic applications. However, the wide range of CDT measurement procedures has resulted in lack of uniform results and reference limits, and hampered comparison of results. In 2005, the IFCC therefore founded a special working group (WG) aiming for standardisation of CDT measurement. This review summarises the history of CDT and the actions taken by the WG-CDT. Initial steps included the definition of the measurand (serum disialotransferrin to total transferrin fraction expressed in %), and the determination of a well-defined anion-exchange HPLC procedure as the candidate reference measurement procedure (cRMP). Subsequent achievements were the establishment of a network of reference laboratories to perform the cRMP, setting a reference interval, and development of a reference material based on human serum for which the laboratory network assign values. Using a set of reference materials for calibration allowed for achieving equivalence of results of all present CDT measurement procedures. The final steps of the WG-CDT have been a full validation of the cRMP to make it an IFCC approved RMP, and providing guidance for international standardisation of all CDT measurement procedures.

Modulation of human motor cortex excitability by single doses of amantadine.

Amantadine-sulfate has been used for several decades to treat acute influenza A, Parkinson's disease (PD), and acute or chronic drug-induced dyskinesia. Several mechanisms of actions detected in vivo/in vitro including N-methyl-D-aspartate (NMDA)-receptor antagonism, blockage of potassium channels, dopamine receptor agonism, enhancement of noradrenergic release, and anticholinergic effects have been described. We used transcranial magnetic stimulation (TMS) to evaluate the effect of single doses of amantadine on human motor cortex excitability in normal subjects. Using a double-blind, placebo-controlled, crossover study design, motor thresholds, recruitment curves, cortical stimulation-induced silent period (CSP), short intracortical inhibition (ICI), intracortical facilitation (ICF), and late inhibition (L-ICI) in 14 healthy subjects were investigated after oral doses of 50 and 100 mg amantadine with single and paired pulse TMS paradigms. Spinal cord excitability was investigated by distal latencies and M-amplitudes of the abductor digiti minimi muscle. After intake of amantadine, a significant dose-dependent decrease of ICF was noticed as well as a significant increase of L-ICI as compared to placebo. The effect on ICF and L-ICI significantly correlated with amantadine serum levels. ICI was slightly increased after amantadine intake, but the effect failed to be significant. Furthermore, amantadine had no significant effects on motor thresholds, MEP recruitment curves, CSP, or peripheral excitability. In conclusion, a low dose of amantadine is sufficient in modulating human motor cortex excitability. The decrease of ICF and increase of L-ICI may reflect glutamatergic modulation or a polysynaptic interaction of glutamatergic and GABA-ergic circuits. Although amantadine has several mechanisms of action, the NMDA-receptor antagonism seems to be the most relevant effect on cortical excitability. As L-ICI can be influenced by this type of drug, it may be an interesting parameter for studies of motor learning and use-dependent plasticity.

Primary biliary cirrhosis is not a clinical condition for increased carbohydrate-deficient transferrin: experience with four independent CDT analysis methods.

BACKGROUND: Primary biliary cirrhosis (PBC) is considered as an important cause for increased carbohydrate-deficient transferrin (CDT). The underlying pathomechanism is difficult to explain by the pathogenesis and/or consequences of PBC. We tested whether PBC causes increased CDT results with current CDT analysis methods and, if so, whether this depends on the CDT analysis principle. METHODS: 48 serum samples from PBC patients were analyzed by HPLC, microcolumn CDT and non-CDT fractionation followed by a turbidimetric immunoassay, particle-enhanced immunonephelometry with monoclonal CDT antibodies, and capillary electrophoresis. The test-specific decision limits were used for categorization of the CDT analysis results into normal and increased values. RESULTS: HPLC: 47 normal/1 increased, microcolumn+TIA: 46 normal/2 increased, particle-enhanced immunonephelometry: 41 normal/7 increased, capillary electrophoresis: 48 normal CDT results. After combining an immunological CDT test (microcolumn+TIA or particle-enhanced immunonephelometry) as the screening method with a physico-chemical CDT test (HPLC or electrophoresis) as the confirmatory method, 1 case remained with increased CDT values by the screening (value 2.6%, cut-off 2.5%, particle-enhanced immunonephelometry) and confirmatory (value 1.8%, cut-off 1.75%, HPLC) analysis. CONCLUSIONS: PBC should no longer be overstressed as an important cause for false-positive CDT results regarding chronic alcohol abuse. In the presence of odd CDT results, PBC should be considered in the anamnestic exploration. However, PBC is not by itself a cause for increased CDT values.

Argininosuccinate lyase deficiency (ASL) and carbohydrate-deficient transferrin (CDT): experience with four independent CDT analysis methods--misleading results given by the %CDT TIA assay.

BACKGROUND: Chronic liver disease can cause false-positive carbohydrate-deficient transferrin (CDT) results mimicking chronic alcohol abuse. We tested whether argininosuccinate lyase deficiency (ASL), a genetic disorder of the urea cycle with hepatomegaly and biochemical hepatitis, causes increased CDT results and whether this depends on the analytical method. METHODS: Seven serum samples from four ASL patients without alcohol abuse were analyzed by capillary electrophoresis, HPLC, particle-enhanced immunonephelometry with monoclonal CDT antibodies, and microcolumn CDT and non-CDT fractionation followed by a turbidimetric immunoassay with transferrin antibodies (%CDT TIA). RESULTS: Increased CDT results (two out of four patients or five out of seven samples) were obtained by the %CDT TIA assay, but not by the remaining three CDT tests. The corresponding serum samples showed increased fractions of trisialotransferrin by HPLC (as the IFCC reference method for CDT analysis). One sample contained an elevated trisialotransferrin but a normal CDT also in the %CDT TIA test. One patient had a normal trisialotransferrin and a normal CDT as assayed by each of the four CDT methods. CONCLUSIONS: Argininosuccinate lyase deficiency is not itself a cause for increased CDT values. Increased fractions of trisialotransferrin in ASL patients appear to interfere with CDT analysis by the %CDT TIA assay. This can give false-positive CDT results. Since this can appear not only in ASL patients, microcolumn CDT and non-CDT fractionation followed by a turbidimetric immunoassay using transferrin but not CDT antibodies by the %CDT TIA assay should no longer be used for CDT measurement without confirmatory analysis by HPLC or capillary electrophoresis.

Excessive urinary excretion of isopropyl glucuronide after isopropanol abuse.

BACKGROUND: Ethyl glucuronide (EtG) in urine is considered a marker of alcohol consumption. We present a case of a false-positive immunological EtG screening result due to excessive isopropyl glucuronide excretion in urine of an alcohol-dependent patient with a history of industrial cleaning fluid abuse. METHODS: EtG screening was done with the Microgenics DRI EtG enzyme immunoassay on a Beckman Coulter AU680 analyzer according to the testkit instructions. Confirmatory analysis was done by LC-MS/MS for EtG, 1-propyl (syn. n-propyl), 2-propyl (syn. isopropyl), 1-butyl, 2-butyl, and tert-butyl glucuronide. Both methods were validated according to the Guidelines of the Society of Toxicological and Forensic Chemistry (GTFCh, Germany). RESULTS: EtG screening by immunoassay was positive, approx. 860mg/L or approx. 1540mg/g creatinine (forensic cut-off 0.1mg/L, clinical cut-off 0.5mg/L). LC-MS/MS confirmatory analysis was negative for EtG (<0.05mg/L; forensic cut-off 0.1mg/L), but strongly positive for 2-propyl glucuronide (approx. 1100mg/L or 2000mg/g creatinine; cut-off 0.1mg/L). 1-propyl, 1-butyl, and tert-butyl glucuronide were negative (<0.05mg/L; cut-off 0.1mg/L), 2-butyl glucuronide was 0.1mg/L (cut-off 0.1mg/L). CONCLUSION: Consumption of household and industrial chemicals with short chain aliphatic alcohols should be considered a rare but potential source of false-positive EtG immunoassay results. Glucuronides from frequently used short chain aliphatic alcohols, like 1-propanol (syn. n-propanol) and 2-propanol (syn. isopropanol) as the most important disinfectant components, should be included into EtG confirmatory analysis. This will be helpful not only for the assessment of the source for remarkable EtG immunoassay results, it can also contribute to a more specific diagnosis in cases with suspected intoxication by consumer or industrial chemical products. Excessive urinary 2-propyl glucuronide (syn. isopropyl glucuronide) concentrations should be considered a marker of isopropanol intoxication.

Standardisation and use of the alcohol biomarker carbohydrate-deficient transferrin (CDT).

Carbohydrate-deficient transferrin (CDT) is a glycoform profile of serum transferrin that increases in response to sustained high alcohol intake and over the last decades has become an important alcohol biomarker with clinical and forensic applications. However, the wide range of CDT measurement procedures has resulted in lack of uniform results and reference limits, and hampered comparison of results. In 2005, the IFCC therefore founded a special working group (WG) aiming for standardisation of CDT measurement. This review summarises the history of CDT and the actions taken by the WG-CDT. Initial steps included the definition of the measurand (serum disialotransferrin to total transferrin fraction expressed in %), and the determination of a well-defined anion-exchange HPLC procedure as the candidate reference measurement procedure (cRMP). Subsequent achievements were the establishment of a network of reference laboratories to perform the cRMP, setting a reference interval, and development of a reference material based on human serum for which the laboratory network assign values. Using a set of reference materials for calibration allowed for achieving equivalence of results of all present CDT measurement procedures. The final steps of the WG-CDT have been a full validation of the cRMP to make it an IFCC approved RMP, and providing guidance for international standardisation of all CDT measurement procedures.

Determination of serum amantadine by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Amantadine (1-adamantylamine) is used for treatment of influenza, hepatitis C, parkinsonism, and multiple sclerosis. Current amantadine analysis by HPLC or gas chromatography (GC) requires a laborious sample pretreatment with extraction and/or derivatization steps. We established an LC-MS/MS method without protein precipitation, centrifugation, extraction and derivatization steps. MATERIAL AND METHODS: 50 microl sample+50 microl of 0.4 mg/l 1-(1-adamantyl)pyridinium bromide as internal standard+1000 microl water (96-well plate). Of this 25 microl+500 microl water (96-well plate; final serum dilution 1:462). LC-MS/MS: Surveyor MS pump, Autosampler, triple-quadrupole TSQ Quantum mass spectrometer (Thermo Electron). Autosampling: 2 microl of each sample. Chromatography: isocratic water/acetonitrile (60/40 v/v) with 5 g/l formic acid, flow rate 0.2 ml/min, run time 3 min, Phenomenex Luna C8(2) (100 x 2.0 mm (i.d.); 3-microm bead size) column. Mass spectrometry: electrospray atmospheric pressure ionization, positive ion and selective reaction monitoring mode, ion transitions m/z 152.0-->135.1 (at 22 eV amantadine) and 214.1-->135.1 (at 26 eV internal standard). RESULTS: Calibration curves were constructed with spiked serum samples (amantadine 50-1000 microg/l, r>0.99). No carry over (5000 microg/l). No ion suppression with retention times similar to those of amantadine (1.8 min) and the internal standard (2.1 min). Detection limit 20 mg/l, linearity 20-5000 mg/l, intra-assay/inter-assay CV<6%/<8%, recovery 99-101%. Method comparison: LC-MS/MS=1.23 x GC-45 (Passing-Bablok regression). No significant bias between GC and LC-MS/MS (Bland-Altman plot). CONCLUSION: We consider the sample pretreatment without deproteination, derivatization and centrifugation steps and the specificity of the tandem mass spectrometry as the most important points of our amantadine analysis method.

Forensic analysis of carbohydrate-deficient transferrin (CDT): implementation of a screening and confirmatory analysis concept is hampered by the lack of CDT isoform standards.

The aim of the study was to test combinations of commercially available carbohydrate-deficient transferrin (CDT) assays for their usefulness as screening and confirmatory CDT analysis systems. A set of 292 serum samples from routine CDT analysis was analyzed by two assays based on anion-exchanger microcolumn CDT and non-CDT fractionation followed by a turbidimetric immunoassay (ChronAlcoI.D. and %CDT TIA) and a high-performance liquid chromatography with on-line sample preparation (ClinRep CDT on-line). The CDT analysis results were divided into four groups based on the test-specific borderlines of the compared methods: NN with negative CDT results by both tests, PN with positive screening but negative confirmation results, NP with negative screening and positive confirmation results, and PP with positive results by both tests. Regardless of the test combination and whether applying the lower or upper limits of the borderlines, approximately one-third of contradictory (positive screening and negative confirmation or vice versa corresponding to groups PN and NP) were obtained. This was not due to analytical outliers (only 6 of 292 serum samples). Indeed, parametric and non-parametric ANOVA analysis pointed to different calibrations and/or recoveries of the three CDT assays. Our data give again evidence for the urgent need of an international CDT isoform standard material. At this time, we cannot recommend a combination of the three tests for screening and confirmatory analysis in forensic CDT testing.

Inhalation but not transdermal resorption of hand sanitizer ethanol causes positive ethyl glucuronide findings in urine.

BACKGROUND: Ethyl glucuronide (EtG) in urine is considered a specific marker of recent ethanol consumption. There is an ongoing debate about whether inhalation or transdermal resorption of sanitizer ethanol is the underlying cause for positive EtG findings after hand disinfection. METHODS: Desderman(®) pure (Schülke & Mayr GmbH, Norderstedt) with 78.2g 96% (v/v) ethanol/100g and approx. 10% 2-propanol was used for multiple hand disinfection without and under an exhauster. Simulating a common working day in a clinic, 5 co-workers of our lab used the sanitizer 32 fold within 8h and 2 persons were merely exposed to the sanitizer vapor but without any dermal sanitizer contact. Any additional ethanol intake or exposition was reliably excluded. Spot urine was collected at baseline, after 1, 2, 4, 6 … 14, and finally 24h after the first sanitizer use. A validated LC-MS/MS was used for MRM and MS(3) of EtG and qualitative analyses of ethyl sulfate and 2-propyl glucuronide. RESULTS: Multiple hand disinfection caused positive EtG findings of up to 2.1mg/L or 1.7mg/g creatinine in 4 out of 5 test persons and even of 0.6mg/L or 0.8mg/g for 2 controls which were merely exposed to the sanitizer vapor but without any sanitizer contact. EtG results between the clinical (0.5mg/g) and the forensic (0.1mg/g) cut-off were obtained even 6h after the last sanitizer exposition. An exhauster prevented the sanitizer vapor inhalation and reduced the EtG excretion to mostly below the detection limit of 0.02mg/g. The maximum value was 0.09mg/g. Ethyl sulfate and 2-propyl glucuronide (2-PpG) were detectable only in the EtG positive samples. 2-PpG is a metabolite of 2-propanol, which is quite frequently used in disinfectants. Thus, the detection of this substance can be used in cases of odd EtG results as an indicator of (unintended) sanitizer exposition. CONCLUSION: Ethanol from hand sanitizers is predominantly incorporated by the respiratory tract but not via the skin. It can cause a distinct ethyl glucuronide excretion and thus analytically true-positive but forensically false-positive EtG findings in the urine of ethanol abstaining persons. Since accidental ethanol inhalation can occur quite frequently in the working place or even private household, such a situation should always be considered when EtG is used as a marker of recent ethanol consumption.

Cross-reaction of propyl and butyl alcohol glucuronides with an ethyl glucuronide enzyme immunoassay.

BACKGROUND: Ethyl glucuronide (EtG) in urine is considered a marker of recent alcohol consumption. Using immunoassays for EtG screening without confirmatory analysis bears a risk of getting false-positives as shown for trichloroethyl glucuronide from chloral hydrate medication and 1-propyl glucuronide from propanol-based hand disinfection. The aim of the study was to check whether glucuronides of frequently used aliphatic short chain alcohols aside from EtG and 1-propyl glucuronide can cross-react with the DRI(®) Ethyl Glucuronide Assay. METHODS: Aliquots of EtG-free urine were individually spiked with methyl ß-D-glucuronide, 1-propyl ß-D-glucuronide, 2-propyl ß-D-glucuronide, 1-butyl ß-D-glucuronide, 2-butyl ß-D-glucuronide, and tert-butyl ß-D-glucuronide. To check the response rate of the DRI(®) Ethyl Glucuronide Assay to its target analyte, EtG was also added to a native EtG-free urine sample. The spiked alcohol glucuronide concentrations (seven levels up to 10mg/L) and the DRI(®) Ethyl Glucuronide Assay results were evaluated by Passing-Bablok regression analysis. The 95% confidence interval ranges for the slope of the regression function were considered a measure of cross-reaction of the individual alcohol glucuronides with the enzyme immunoassay. RESULTS: 2-Propyl glucuronide showed a cross-reactivity of 69-84% at the 95% probability level, methyl glucuronide, 1-propyl glucuronide, and 1- and 2-butyl glucuronide of 4-9%, and tert-butyl glucuronide almost no cross-reactivity. The response rate for EtG was 87-94% at the 95% probability level. CONCLUSION: The DRI(®) Ethyl Glucuronide Assay shows cross-reaction rates with aliphatic short chain alcohol glucuronides aside from EtG which bear a risk of getting false-positives regarding ethanol consumption. Mass spectrometric detection of EtG is mandatory for confirmation of positive immunological EtG screenings.

Ethyl glucuronide identified in commercial hair tonics.

BACKGROUND: Ethyl glucuronide (EtG) in hair is considered as a specific marker of ethanol consumption. Prompted by a report of positive EtG hair testings due to hair treatment with an EtG containing hair lotion, commercially available herbal hair tonics from supermarkets, drug-stores, and health food stores were analyzed for the presence of EtG and ethyl sulfate (EtS). METHODS: LC-MS/MS (QTRAP 5500 mass spectrometer) was done in multiple reaction monitoring (MRM), enhanced product ion (EPI) and MS(3) mode. The lower limit of quantitation was 0.05 mg/L for EtG and the cut-off for the detection of EtS 0.01 mg/L. RESULTS: Altogether 11 hair tonics from 8 manufacturers were tested, with 1 product in 3 different lots. EtG ranged between 0.07 and 1.06 mg/L (7 products from 4 manufacturers) and was almost identical in the 3 lots of 1 product (1.01-1.06 mg/L). EtS was found in 3 out of the 11 hair tonics. CONCLUSIONS: EtG is quite frequently present in commercially available herbal hair tonics. Using EtG in hair as a marker of alcohol (ab)use, one has to consider external sources of EtG and has to assess the use of hair care products, esp. if the patient denies any ethanol intake. Whether EtS is a more reliable alcohol (ab)use marker, as sometimes discussed, should be critically assessed against the background of its broad use in large amounts in industrial chemistry.

Functional genomics of pain in analgesic drug development and therapy.

Advances in genomic research have led to the clarification of the detailed involvement of gene products in biological pathways and these are being increasingly exploited in strategies for drug discovery and repurposing. Concomitant developments in informatics have resulted in the acquisition of complex gene information through the application of computational analysis of molecular interaction networks. This approach enables the acquired knowledge on hundreds of genes to be used to view molecular disease mechanisms from a genetic point of view. By analyzing 410 genes which control the complex process of pain, we show by computational analysis, based on functional annotations to pain-related genes, that 12 clearly circumscribed functional areas are essential for pain perception and thus for analgesic drug development. The genetics perspective revealed that future development strategies should focus on substances modulating intracellular signal transduction, ion transport and anatomical structure development. These processes are involved in the genetic-based absence of pain and therefore, provide promising fields for curative or preventive treatments. In contrast, interactions with G-protein coupled receptor pathways seem merely to provide symptomatic, not preventative relief of pain. In addition, biological functions accessed either by analgesic drugs or microRNAs suggest that synergistic therapies may be a future direction for drug development. With modern computational functional genomics, it is possible to exploit genetic information from increasingly available data sets on complex diseases, such as pain, and offers a new insight into drug development and therapy which is complementary to pathway-centered approaches.

False-positive ethyl glucuronide immunoassay screening caused by a propyl alcohol-based hand sanitizer.

BACKGROUND: Urine ethyl glucuronide (EtG) is considered as a specific marker of recent ethanol consumption. We describe false-positive DRI(®) EIA EtG enzyme immunoassay results caused by propyl glucuronides in urine after using a propanol-based hand sanitizer. METHODS: EtG screening was done with the DRI(®) EIA EtG assay (Microgenics), using a cut-off of 0.5 mg/L as recommended by the manufacturer and of 0.1 mg/L as demanded by the German Regulations for Reissuing Drivers Licenses. Confirmatory EtG analysis was done with the ClinMass(®) EtG LC-MS/MS testkit (Recipe), extended by the mass transitions 235.1→75.1, 235.1→85.1, and 235.1→113.1 for the detection of the 1- and 2-propyl glucuronides. Self-experiments were done by staff members of our lab (n=7), using 3 mL Sterillium(®) Classic Pure (30 g/100 g 1-propanol and 45 g/100 g 2-propanol) for hand sanitation every quarter of an hour for 8 h according to DIN EN 1500:2011-05 with and without an exhauster and by passive inhalation of the sanitizer vapor. Spot urine samples were taken immediately before and up to 24 h after the first sanitizer use. RESULTS: False-positive immunoassay results of up to 4 mg/L or 2.3 mg/g creatinine were obtained after normal use of the sanitizer and also after passive inhalation of the sanitizer vapor (up to 0.89 mg/L or 0.61 mg/g). Immunoassay results were positive even after 4-fold use of the sanitizer (up to 0.14 mg/L or 0.38 mg/g) and up to 6 h after the last sanitizer contact (maximum 0.63 mg/L and 0.33 mg/g for sanitizer users and 0.25 mg/g after passive inhalation). Spiking of EtG-free urine with 1-propyl glucuronide (Athena Environmental Sciences) between 0.05 and 10 mg/L clearly demonstrated a cross reaction of the immunoassay of approx. 10% as compared to EtG. LC-MS/MS of urines with a positive immunoassay EtG result did not show EtG signals, but distinct signals of 1-propyl glucuronide (n-propyl glucuronide) and 2-propyl glucuronide (iso-propyl glucuronide). An exhauster effectively prevented the inhalation of the sanitizer vapor, the formation of propyl glucuronides and thus false-positive DRI(®) EIA EtG screening results, proving that propyl alcohols are almost exclusively taken up by respiration. CONCLUSIONS: The widespread use of propanol-containing products such as hand sanitizers may lead to sufficient uptake of propyl alcohols and excretion of significant amounts of propyl glucuronides to yield false-positive DRI(®) EIA EtG screening results. Thus, positive EtG immunoassay results have to be controlled by mass-spectrometry, in clinical cases at least if ethanol intake is denied by the patient.