An enzymatic method to determine γ-hydroxybutyric acid in serum and urine.

BACKGROUND: Gamma-hydroxybutyric acid (GHB) has become one of the most dangerous illicit drugs of abuse today. It is used as a recreational and date rape drug because of its depressant effect on the central nervous system, which may cause euphoria, amnesia, respiratory arrest, and coma. There is an urgent need for a simple, easy-to-use assay for GHB determination in urine and blood. In this article, a rapid enzymatic assay adapted to clinical chemistry analyzers for the detection of GHB is presented. METHODS: The described GHB enzymatic assay is based on a recombinant GHB dehydrogenase. The full validation of the assay was performed on a Konelab 30 analyzer (Thermo Fisher Scientific). RESULTS: The analytical sensitivity was <1.5 mg/L, whereas the functional sensitivity was 4.5 mg/L in serum and 2.8 mg/L in urine. The total imprecision coefficient of variation (CV) was <9.8% in serum and <7.9% in urine. The within-run imprecision showed a CV of <3.8% in serum and <4.6% in urine. The assay was linear within the range 5-250 mg/L. Mean recoveries were 109% in serum and 105% in urine. No cross-reactivity was observed for tested GHB analogues and precursors. Comparison of GHB-positive samples showed an excellent correlation with ion chromatography, gas chromatography-mass spectrometry, and liquid chromatography associated to tandem mass spectrometry. Except for ethanol, no substantial interference from serum constituents and some drugs was observed. CONCLUSIONS: This automated GHB assay is fully quantitative and allows the accurate measurement of GHB in serum and urine. It can be used as a rapid screening assay for the determination of GHB in intoxicated or overdosed patients.

An automated screening method for drugs and toxic compounds in human serum and urine using liquid chromatography-tandem mass spectrometry.

A fully automated screening using liquid chromatography-mass spectrometric method applying data-dependent acquisition was developed to identify toxicologically relevant substances in serum and urine. A library including more than 405 spectra of about 365 compounds (main drugs and important metabolites) was established. An easy to use program was created to automate and accelerate library search. Drugs were identified based on their relative retention times, molecular ions and fragment ions. Limits of detection were tested with 100 of the 365 compounds the majority of these were lower than 100µg/l (67%). The developed LC-MS-MS system seems to be a valuable alternative to other general unknown screening methods allowing fast and specific identification of drugs in serum and urine samples.

Development and characterization of an enzymatic method for the rapid determination of gamma hydroxybutyric acid.

Gamma hydroxybutyric acid (GHB) is a regulated therapeutic drug, which naturally occurs in mammalian brain tissues as an intermediate of the GABA (gamma aminobutyric acid) neurotransmitter metabolism. The increasing misuse of GHB as a narcotic or abusing drug in recent years calls for the development of a simple and rapid screening method as an alternative to the currently available, technically demanding diagnostic methods. We have developed a rapid enzymatic assay based on the GHB dehydrogenase of Ralstonia eutropha. The enzyme is expressed as a recombinant protein in Escherichia coli and characterized in terms of reaction mechanism and kinetic parameters for the catalysis of conversion of GHB into succinic semialdehyde (SSA). The concomitant NADH production enables spectrophotometric monitoring of the reaction and the quantification of GHB in physiological fluids depending on initial velocities. We have tested a panel of twelve serum and urine samples containing GHB concentrations from 0.0 to 2.1 mmol/L. GHB dehydrogenase activity obeys a non classical bi bi ping pong mechanism exhibiting substrate inhibition by NAD+. With an optimal NAD+ concentration of 3.7 mmol/L in the reaction, the enzyme yields a K(M) of 1.0 mmol/L for GHB and a Vmax of 3.37 mmol/min/mg. The assay shows a linear standard curve from 0.1 to at least 1 mmol/L of GHB. Spiking experiments result in mean recoveries of 92% for urine and 114% for serum, respectively. The comparison to an ion chromatographic reference method exhibits a mean difference of 10% divergence from the target values in urine and 9% in serum, respectively.

Determination of gamma-hydroxybutyric acid in clinical samples using capillary electrophoresis with contactless conductivity detection.

A method for the determination of gamma-hydroxybutyric acid (GHB) in urine and serum samples with capillary electrophoresis using capacitively coupled contactless conductivity detection (CE-C(4)D) was developed. The optimized separation buffer consisting of 20 mM of arginine, 10 mM of maleic acid and 30 microM of cetyltrimethylammonium bromide (CTAB) contained 5 mM vancomycin to facilitate the separation of gamma-hydroxybutyric acid from beta-hydroxybutyric acid (BHB), which is also present in clinical samples. The detection limits in the clinical samples were found to be about 2 microg/ml, which is well below the required sensitivity for the recognition of overdosage and adequate for the determination of endogenous concentrations in urine. The determination of GHB in both types of samples was carried out directly after a fourfold dilution without requiring any derivatization or extraction procedures.

Sensitivity and specificity of urinary cannabinoid detection with two immunoassays after controlled oral administration of cannabinoids to humans.

For forensic and clinical toxicologic purposes, cannabis consumption is screened using easy-to-handle immunoassays. The sensitivity and specificity of these immunoassays have not yet been established in samples from volunteers receiving oral synthetic tetrahydrocannabinol or cannabis extracts using liquid chromatography-mass spectrometry/mass spectrometry as the reference method. Urine samples were collected in an open, randomized, single-center, three-period crossover study including 18 healthy male volunteers given either 20 mg synthetic tetrahydrocannabinol (Marinol) as a control substance or five different types of Cannabis sativa extracts.

Undetectable phenytoin serum levels by an automated particle-enhanced turbidimetric inhibition immunoassay in a patient with monoclonal IgM lambda.

INTRODUCTION: Phenytoin is a drug used for the treatment of different types of seizures. Its variable pharmacokinetics, mainly a consequence of variable bioavailability, saturable protein binding and saturable hepatic metabolism, predisposes the drug to therapeutic drug monitoring. Several methods to analyze the drug in serum exist with immunoassays being the method of choice for routine measurements. Immunoassays are specific and sensitive, but cross-reactivity, possibly leading to erroneous serum levels, is a concern. We report a patient with falsely undetectable phenytoin serum levels. CASE REPORT: This 73-year old woman was treated with intravenous phenytoin due to epilepsia partialis continua in the context of a bilateral cerebrovascular insult. Anamnestically, a chronic lymphatic leukemia was known. In this patient, serum phenytoin levels became only detectable by the particle enhanced turbidimetric inhibition immunoassay used after precipitation of serum proteins. Protein electrophoresis revealed a monoclonal immunoglobulin, identified as IgMlambda. With other methods such as HPLC and fluorescence depolarization immunoassay, phenytoin was detectable. CONCLUSION: We propose interference between the monoclonal IgMlambda and/or other serum proteins and the particle-enhanced turbidimetric inhibition immunoassay, rendering phenytoin falsely undetectable in samples of this patient. In such patients, alternative methods such as HPLC should be used to prevent dosage errors.

Medical and economic long-term effects of B-type natriuretic peptide testing in patients with acute dyspnea.

BACKGROUND: The objective of this prospective study was to assess the medical and economic long-term effects of using B-type natriuretic peptide (BNP) concentrations in the management of patients with acute dyspnea. METHODS: We performed follow-up analysis of the B-Type Natriuretic Peptide for Acute Shortness of Breath Evaluation, a randomized study including 452 patients who presented to the emergency department with acute dyspnea. Participants were randomly assigned to a diagnostic strategy involving the rapid measurement of BNP concentrations (n = 225) or standard assessment (n = 227). Mortality was assessed at 720 days, morbidity and economic data at 360 days. RESULTS: BNP testing induced several important changes in initial patient management, including a reduction in the initial hospital admission rate, the use of intensive care, and initial time to discharge. At 720 days, 172 deaths had occurred. Cumulative all-cause 720-day mortality was not different between the BNP group (37%) and the control group (36%, P = 0.6). Morbidity as reflected by days spent in-hospital at 360 days was significantly lower in the BNP group [median 12 days ([interquartile range 2-28 days)] compared with the control group [median 16 (7-32)] days, P = 0.025]. Functional status was similar in both groups. Economic outcome as quantified by total treatment cost at 360 days was significantly improved in the BNP group (mean 10,144 dollars vs 12,748 dollars in the control group, P = 0.008). CONCLUSIONS: Rapid BNP testing in patients with acute dyspnea has no effect on long-term mortality. However, morbidity as quantified by days spent in-hospital and economic outcome are still improved at 360 days.

Development of a LC/MS/MS method for the analysis of cannabinoids in human EDTA-plasma and urine after small doses of Cannabis sativa extracts.

A novel high-performance liquid chromatographic separation method with tandem-mass spectrometry detection was developed for the simultaneous determination of Delta(9)-tetrahydrocannabinol (THC) and its major metabolites 11-hydroxy-Delta(9)-tetrahydrocannabinol (11-OH-THC) and 11-nor-Delta(9)-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) as well as the components cannabidiol (CBD) and cannabinol (CBN) in human EDTA-plasma and urine. Run time was 25 min. Lower limit of quantification was 0.2 ng/ml. The coefficients of variation of all inter- and intra-assay determinations were between 1.3 and 15.5%. The method was successfully applied to the determination of cannabinoids in human plasma and human urine after administration of Delta(9)-tetrahydrocannabinol or Cannabis sativa extracts.

Evaluation of a microparticle enzyme immunoassay for the measurement of sirolimus in whole blood (Abbott IMx sirolimus).

An immunoassay of sirolimus based on the microparticle enzyme immunoassay (MEIA) principle was evaluated on 105 whole blood samples (EDTA) drawn from a population of renal (n = 38) and hepatic or heart (n = 3) transplant patients. Each blood sample was analyzed simultaneously by MEIA and by a liquid chromatography mass spectrometry (LC-MS) method. The statistical analysis according to Passing-Bablok (x= LCMS, y= mean of two measurements of the same samples on IMx) produced the following results: Spearman r value = 0.9651, y(MEIA) = 0.99x (LCMS) - 0.26 microg/l. The analytical performance of the MEIA method showed a CV < or = 10% and a lower limit of quantification of 1.5 microg/l, which are acceptable for routine clinical monitoring. In conclusion, the MEIA method has shown robust, stable and reproducible features with an excellent correlation with the LC-MS method.

Survival of severe amlodipine intoxication due to medical intensive care.

We report the case of attempted suicide with amlodipine, chlorthalidone and mefenamic acid and subsequent medical intensive care measures which resulted in total recovery of a 42-year-old male. After admission to the medical intensive care unit the intoxicated patient was deeply hypotensive and needed fluid replacement, dobutamine and norepinephrine. Additionally insulin and calcium gluconate were given. Since hypotension persisted and the patient developed oliguria, terlipressin was applied and finally showed an effect on blood pressure and on urinary output. A volume overload of 7 L in the first 24 h resulted in a pulmonary edema. The patient was started on non-invasive ventilation with continuous positive airway pressure (CPAP) and frusemide was added to the therapy with good success. Quantitative determination of amlodipine in plasma samples was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The highest amlodipine concentrations was measured in the plasma sample collected approximately 8 h after ingestion of the drug, and was 393 microg/L. Four days later, it was possible to stop the treatment with catecholamines, at that time the amlodipine plasma concentration had declined to 132 microg/L, still tenfold higher than therapeutic (5-18 microg/L). Elimination half-life of amlodipine is approximately 55 h. After 6 days in the intensive care unit the patient was transferred to psychiatric treatment. Intensive care management and plasma levels in this intoxication case are compared to data from literature on other cases.

Cost-effectiveness of B-type natriuretic peptide testing in patients with acute dyspnea.

BACKGROUND: B-type natriuretic peptide (BNP) is a quantitative marker of heart failure that seems to be helpful in its diagnosis. METHODS: We performed a prospective randomized study (B-Type Natriuretic Peptide for Acute Shortness of Breath Evaluation) including 452 patients who presented to the emergency department with acute dyspnea to estimate the long-term cost-effectiveness of BNP guidance. Participants were randomly assigned to a diagnostic strategy involving the measurement of BNP levels (n = 225) or assessment in a standard manner (n = 227). Nonparametric bootstrapping was used to estimate the distribution of incremental costs and effects on the cost-effectiveness plane during 180 days of follow-up. RESULTS: Testing of BNP induced several important changes in management of dyspnea, including a reduction in the initial hospital admission rate, the use of intensive care, and total days in the hospital at 180 days (median, 10 days [interquartile range, 2-24 days] in the BNP group vs 14 days [interquartile range, 6-27 days] in the control group; P = .005). At 180 days, all-cause mortality was 20% in the BNP group and 23% in the control group (P = .42). Total treatment cost was significantly reduced in the BNP group (7930 dollars vs 10,503 dollars in the control group; P = .004). Analysis of incremental 180-day cost-effectiveness showed that BNP guidance resulted in lower mortality and lower cost in 80.6%, in higher mortality and lower cost in 19.3%, and in higher or lower mortality and higher cost in less than 0.1% each. Results were robust to changes in most variables but sensitive to changes in rehospitalization with BNP guidance. CONCLUSION: Testing of BNP is cost-effective in patients with acute dyspnea.

Use of B-type natriuretic peptide in the management of acute dyspnea in patients with pulmonary disease.

BACKGROUND: In patients with pulmonary disease, it is often challenging to distinguish exacerbated pulmonary disease from congestive heart failure (CHF). The impact of B-type natriuretic peptide (BNP) measurements on the management of patients with pulmonary disease and acute dyspnea remains to be defined. METHODS: This study evaluated the subgroup of 226 patients with a history of pulmonary disease included in the BASEL Study. Patients were randomly assigned to a diagnostic strategy with (n = 119, BNP group) or without (n = 107, clinical group) the use of BNP levels provided by a rapid bedside assay. Time to discharge and total cost of treatment were recorded as the primary end points. RESULTS: Baseline characteristics were similar in patients assigned to the BNP and control groups. Comorbidity was extensive, including coronary artery disease and hypertension in half of patients. The primary discharge diagnosis was CHF and exacerbated obstructive pulmonary disease in 39% and 33%, respectively. The use of BNP levels significantly reduced the need for hospital admission (81% vs 91%, P = .034). Median time to discharge was 9.0 days in the BNP group as compared with 12.0 days (P = .001) in the clinical group. Median total cost of treatment was $4841 in the BNP group as compared with $5671 in the clinical group (P = .008). Inhospital mortality was 8% in both groups. CONCLUSIONS: CHF is a major cause of acute dyspnea in patients with a history of pulmonary disease. Used in conjunction with other clinical information, rapid measurement of BNP reduced time to discharge and total treatment cost of these patients.

B-type natriuretic peptide for acute dyspnea in patients with kidney disease: insights from a randomized comparison.

BACKGROUND: B-type natriuretic peptide (BNP) levels are reliably elevated in patients with congestive heart failure (CHF) and therefore helpful in its diagnosis. However, kidney disease results in elevated BNP levels independently of CHF. Accordingly, the impact of kidney disease on the benefit of BNP testing needs to be scrutinized. METHODS: This study evaluated patients with and without kidney disease [glomerular filtration rate (GFR) less than 60 mL/min/1.73 m(2)) presenting with acute dyspnea. A total of 452 consecutive patients (240 with kidney disease and 212 without kidney disease) were randomly assigned to a diagnostic strategy with (BNP group) or without (control group) the use of BNP levels provided by a rapid bedside assay. RESULTS: Patients with kidney disease were older, more often had CHF as the cause of acute dyspnea, and more often died in-hospital or within 30 days as compared to patients without kidney disease. In patients without kidney disease, BNP testing significantly reduced median time to discharge (from 9.5 days to 2.5 days) (P= 0.003) and total cost of treatment (from 7184 dollars to 4151 dollars) (P= 0.004). In contrast, in patients with kidney disease, time to discharge and total cost of treatment were similar in both groups. CONCLUSION: When applying BNP cut-off values without adjustment for the presence of kidney disease, the use of BNP levels does significantly improve the management of patients without kidney disease, but not of those with kidney disease.

Use of B-type natriuretic peptide for the management of women with dyspnea.

In studies with predominately male patients, B-type natriuretic peptide (BNP) levels have been shown to be helpful in the evaluation and management of patients who present with acute dyspnea. The effect of BNP levels on the management of women has not been defined. This study evaluated a predefined subgroup of 190 women included in a prospective randomized controlled study of BNP testing for emergency diagnosis of acute dyspnea. Patients were randomly assigned to a diagnostic strategy with (n = 93, BNP group) or without (n = 97, control group) the use of BNP levels provided by a rapid bedside assay. Women differed significantly from men in baseline characteristics, symptoms, signs, and final discharge diagnoses. The use of BNP levels decreased the need for hospital admission (73% vs 86%, p = 0.034) and intensive care (12% vs 23%, p = 0.048). Median time to discharge was 6 days in the BNP group versus 10 days in the control group (p = 0.023). Total cost of treatment was $4,781 in the BNP group (95% confidence interval 3,854 to 5,708) versus $6,843 in the control group (95% confidence interval 5,611 to 8,074, p = 0.009). In-hospital mortality rates were 4% in the BNP group and 10% in the control group (p = 0.165). Thus, used in conjunction with other clinical information, rapid measurement of BNP decreased time to discharge and total cost of treatment in women who presented with acute dyspnea.

High prevalence of unknown co-medication in hospitalised patients.

OBJECTIVE: Co-medication unknown to the treating physician, including self-medication, may compromise drug safety by increasing the risk of duplicate therapy, drug interactions and adverse drug reactions that are not recognised as such. The aim of the current study was to estimate exposure to unknown co-medication during hospitalisation by performing an analytical screening for a broad range of drugs and drug classes in urine of patients admitted to a general internal medicine ward. METHODS: Urine samples of 44 patients were analysed with REMEDiHS (high-performance liquid chromatography) and six different immunoassays. Positive results were compared with drug history and documented drug prescription. If appropriate, gas chromatographic-mass spectrometric confirmatory analyses were performed on drugs classified at least once as possible unknown co-medication. RESULTS: Nine (20%) of the patients tested positive for a compound detected by two independent analytical methods and 18 (41%) for a compound detected by at least one analytical method. Unknown co-medication consisted mostly of analgesics, benzodiazepines or ranitidine. CONCLUSION: At least one in five patients exhibits at least once during hospitalisation exposure to drugs not documented in the patient record, which may compromise patient safety.

Use of B-type natriuretic peptide in the evaluation and management of acute dyspnea.

BACKGROUND: B-type natriuretic peptide levels are higher in patients with congestive heart failure than in patients with dyspnea from other causes. METHODS: We conducted a prospective, randomized, controlled study of 452 patients who presented to the emergency department with acute dyspnea: 225 patients were randomly assigned to a diagnostic strategy involving the measurement of B-type natriuretic peptide levels with the use of a rapid bedside assay, and 227 were assessed in a standard manner. The time to discharge and the total cost of treatment were the primary end points. RESULTS: Base-line demographic and clinical characteristics were well matched between the two groups. The use of B-type natriuretic peptide levels reduced the need for hospitalization and intensive care; 75 percent of patients in the B-type natriuretic peptide group were hospitalized, as compared with 85 percent of patients in the control group (P=0.008), and 15 percent of those in the B-type natriuretic peptide group required intensive care, as compared with 24 percent of those in the control group (P=0.01). The median time to discharge was 8.0 days in the B-type natriuretic peptide group and 11.0 days in the control group (P=0.001). The mean total cost of treatment was 5,410 dollars (95 percent confidence interval, 4,516 dollars to 6,304 dollars) in the B-type natriuretic peptide group, as compared with 7,264 dollars (95 percent confidence interval, 6,301 dollars to 8,227 dollars) in the control group (P=0.006). The respective 30-day mortality rates were 10 percent and 12 percent (P=0.45). CONCLUSIONS: Used in conjunction with other clinical information, rapid measurement of B-type natriuretic peptide in the emergency department improved the evaluation and treatment of patients with acute dyspnea and thereby reduced the time to discharge and the total cost of treatment.

Leptin levels of alcohol abstainers and detoxification patients are not different.

AIMS: Leptin is a cytokine-type peptide hormone, recently implicated as a putative state marker of alcohol use and in craving. Our goal was to evaluate the potential of leptin as a state and trait marker and to rule out the role of current alcohol intoxication on leptin levels. METHODS: Eighteen alcohol withdrawal patients (16 males, 2 females) whose blood contained 202 mg/dl (median) of ethanol at hospitalization, who had a median age of 43.5 years and had consumed 1075 g of ethanol (median) in the last 7 days were included in the study. Leptin was determined in samples at day 1 (when still intoxicated) and day 7 of withdrawal. Expected leptin levels were calculated with a formula. For comparison, 27 blood samples of 18 abstinent persons, matched for gender, age and body mass index were used. Furthermore, mean cell volume, gamma-glutamyl transferase (GGT), blood glucose, cholesterol, triglycerides and body composition (bioimpedance device) were determined. For statistical analysis, SPSS 11 was used. RESULTS: Expected leptin levels were 1.71 ng/ml (median), leptin measured at day 1 was 2.65 ng/ml (median) and 2.85 ng/ml on day 7 for the alcohol withdrawal patients and 2.2 ng/ml (median) for the abstainers. These concentrations were not significantly different. Significant correlations were found between leptin day 1 and expected leptin levels, percentage fat body mass, cigarettes smoked per day, GGT and blood alcohol concentration. CONCLUSIONS: Our preliminary data do not support the hypothesis of leptin as a state or trait marker and suggest only a minor influence of acute intoxication on leptin levels in alcohol detoxification patients.

Windows to the ward: graphically oriented report forms. Presentation of complex, interrelated laboratory data for electrophoresis/immunofixation, cerebrospinal fluid, and urinary protein profiles.

BACKGROUND: Automated laboratory analyzers that mass produce data have been linked to information systems for more than two decades, but little progress has been made in developing more comprehensible report forms. Results are still reported in computer-generated printouts containing hundreds of numbers crowded into columns on each printed page. METHODS: We developed three software applications focusing on the graphic presentation of laboratory results. RESULTS: The first application summarizes data for a patient with a monoclonal gammopathy. The report provides a cumulative graphic presentation of immunofixation/electrophoresis data without any additional interpretation, focuses on a color-coded electrophoresis scan, and records up to 5 years on a single page. The second application deals with cerebrospinal fluid analysis. The report calculates relevant data and graphs the complex relationship between albumin and immunoglobulin results from paired serum and cerebrospinal fluid samples. Manually added interpretive text assures an output comprehensible to clinicians in all specialties. The third application produces a report summarizing quantitatively measured urinary marker protein profiles. The report form is generated by a flexible, completely user-definable knowledge-based system. It calculates numerous ratios and formulae, supports reflex testing, supplies an automated interpretation, and generates a specific graphic signature pattern of the results (MDI LabLink proteinuria differentiation). CONCLUSIONS: Increased clinical demand for graphically oriented report forms 5 years after their introduction has provided evidence that these reports transfer complex laboratory data and results to the clinician more effectively. The highest (more than threefold) increase in demand has been for reports for urinary marker protein profiles that feature a largely self-explanatory graphic signature pattern.

Low biochemical nutritional parameters in acutely ill hospitalized elderly patients with and without stage III to IV pressure ulcers.

BACKGROUND AND AIMS: Pressure ulcers are associated with impaired nutritional status in acutely ill elderly patients. The objective of this study was to establish whether a difference exists between biochemical nutritional parameters in acutely ill elderly with stage III to IV pressure ulcers and a group of acutely ill elderly with no pressure ulcers. METHODS: In a retrospective study we compared 8 biochemical nutritional markers in a group of 22 acutely ill elderly patients consecutively admitted to the geriatric ward who had stage III to IV pressure ulcers (PU group) in addition to their acute illness with a control group of 40 acutely ill elderly patients with no pressure ulcers (NPU group). RESULTS: The PU group compared with the NPU group had significantly lower (p < 0.0001) values of albumin, transferrin, hemoglobin, cholesterol, iron, and zinc (p < 0.0059). Total lymphocyte count was slightly, but not significantly lower in the PU group. In contrast, C-Reactive Protein levels were significantly higher (p < 0.0001) in the PU group compared with the NPU group, indicating a more severe illness in the presence of additional pressure ulcers. CONCLUSIONS: In this study, serum levels of biochemical nutritional parameters in acutely ill elderly patients with stage III to IV pressure ulcers are lower than those of acutely ill elderly subjects with no pressure ulcers, indicating a worse nutritional status of the PU patients. These findings, while not documenting a causal relationship, suggest the need for routine nutritional assessment and support in older patients, especially those with pressure ulcers.