Clinical characteristics do not reliably identify non-adherence in patients with uncontrolled hypertension.

PURPOSE: Chemical adherence testing is a reliable method to assess adherence to antihypertensive drugs. However, it is expensive and has limited availability in clinical practice. To reduce the number and costs of chemical adherence tests, we aimed to develop and validate a clinical screening tool to identify patients with a low probability of non-adherence in patients with uncontrolled hypertension. MATERIALS AND METHODS: In 495 patients with uncontrolled hypertension referred to the University Medical Centre Utrecht (UMCU), the Netherlands, a penalised logistic regression model including seven pre-specified easy-to-measure clinical variables was derived to estimate the probability of non-adherence. Non-adherence was defined as not detecting at least one of the prescribed antihypertensive drugs in plasma or urine. Model performance and test characteristics were evaluated in 240 patients with uncontrolled hypertension referred to the Heartlands Hospital, United Kingdom. RESULTS: Prevalence of non-adherence to antihypertensive drugs was 19% in the UMCU and 44% in the Heartlands Hospital population. After recalibration of the model's intercept, predicted probabilities agreed well with observed frequencies. The c-statistic of the model was 0.63 (95%CI 0.53-0.72). Predicted probability cut-off values of 15%-22.5% prevented testing in 5%-15% of the patients, carrying sensitivities between 97% (64-100) and 90% (80-95), and negative predictive values between 74% (10-99) and 70% (50-85). CONCLUSION: The combination of seven clinical variables is not sufficient to reliably discriminate adherent from non-adherent individuals to safely reduce the number of chemical adherence tests. This emphasises the complex nature of non-adherence behaviour and thus the need for objective chemical adherence tests in patients with uncontrolled hypertension.

A Diagnostic Algorithm for Children with Low Alkaline Phosphatase Activities: Lessons Learned from Laboratory Screening for Hypophosphatasia.

OBJECTIVES: To explore the role of laboratory screening for hypophosphatasia and propose a diagnostic pathway for children with low serum alkaline phosphatase (ALP) activities. STUDY DESIGN: A retrospective hospital-based study over an 8-year period was conducted to identify children younger than 16 years of age with low ALP activity (<100 U/L). Study-positive patients were contacted for repeat sampling, and those with persistently low ALP had plasma pyridoxal-5'-phosphate and urinary phosphoethanolamine measured. RESULTS: Of 323 064 analyzed samples, 1526 had ALP activities <100 U/L. Most patients had transient hypophosphatasemia. Of 50 patients with last-recorded ALP <100 U/L, 32 were excluded given previous ALP >100 U/L. Eighteen were identified as study-positive. Of the 15 surviving children, 13 were traceable. Four had persistently low ALP activity on retesting, of whom 2 had raised pyridoxal-5'-phosphate and phosphoethanolamine concentrations and were subsequently tested for ALPL gene mutations; a 4-year-old asymptomatic girl with a novel homozygous ALPL missense mutation and a 23-year-old female with a heterozygous mutation. There was significant overlap in ALP activities between study-positive and 11 current patients with hypophosphatasia. We propose a diagnostic algorithm for children with low ALP activities based on clinical and biochemical variables. CONCLUSIONS: Patients with persistently low ALP activity require further clinical, biochemical, and radiological assessment for hypophosphatasia, even in the absence of clinical symptoms. The proposed diagnostic algorithm for children with low ALP will facilitate early detection of cases of hypophosphatasia, which, with the availability of enzyme replacement for hypophosphatasia, can be life-saving or avoid years of undiagnosed morbidity.

Cortisone-reductase deficiency associated with heterozygous mutations in 11beta-hydroxysteroid dehydrogenase type 1.

In peripheral target tissues, levels of active glucocorticoid hormones are controlled by 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), a dimeric enzyme that catalyzes the reduction of cortisone to cortisol within the endoplasmic reticulum. Loss of this activity results in a disorder termed cortisone reductase deficiency (CRD), typified by increased cortisol clearance and androgen excess. To date, only mutations in H6PD, which encodes an enzyme supplying cofactor for the reaction, have been identified as the cause of disease. Here we examined the HSD11B1 gene in two cases presenting with biochemical features indicative of a milder form of CRD in whom the H6PD gene was normal. Novel heterozygous mutations (R137C or K187N) were found in the coding sequence of HSD11B1. The R137C mutation disrupts salt bridges at the subunit interface of the 11ß-HSD1 dimer, whereas K187N affects a key active site residue. On expression of the mutants in bacterial and mammalian cells, activity was either abolished (K187N) or greatly reduced (R137C). Expression of either mutant in a bacterial system greatly reduced the yield of soluble protein, suggesting that both mutations interfere with subunit folding or dimer assembly. Simultaneous expression of mutant and WT 11ß-HSD1 in bacterial or mammalian cells, to simulate the heterozygous condition, indicated a marked suppressive effect of the mutants on both the yield and activity of 11ß-HSD1 dimers. Thus, these heterozygous mutations in the HSD11B1 gene have a dominant negative effect on the formation of functional dimers and explain the genetic cause of CRD in these patients.

Broad evidence of xylazine in the UK illicit drug market beyond heroin supplies: Triangulating from toxicology, drug-testing and law enforcement.

BACKGROUND AND AIMS: Xylazine is a non-opioid sedative which has spread rapidly throughout the US illicit drug supply. This study aimed to describe the spread of xylazine throughout the UK illicit drug supply. METHODS: Xylazine detections in human biological samples were collated from toxicology laboratories operating in the United Kingdom with the date, location, case type, xylazine concentration and co-detected drugs (with quantifications where performed) detailed, where permitted, by the corresponding coroner. Drug-testing cases positive for xylazine were collated from the Welsh Emerging Drugs and Identification of Novel Substances (WEDINOS) drug-testing postal service with the date, location, purchase intent and co-detected drugs detailed. Drug seizures made by UK law enforcement were communicated by the Office for Health Improvement and Disparities with the date and location detailed. RESULTS: By the end of August 2023, xylazine was detected in 35 cases from throughout toxicology, drug-testing and drug seizure sources covering England, Scotland and Wales. There were no cases reported from Northern Ireland. Xylazine was detected in biological samples from 16 people. In most cases where full toxicology results were provided, xylazine was detected with heroin and/or a strong opioid (n = nine of 11), but this polydrug use pattern was not evident in all cases (n = two of 11), suggesting a wider circulation of xylazine in the UK illicit drug market beyond heroin supplies. Evidence from WEDINOS supports this claim, as all 14 drug samples (100%) submitted from across the UK contained xylazine; however, in none of these cases was heroin the purchase intent but rather counterfeit prescription medication tablets (n = 11 of 14), tetrahydrocannabinol (THC) vapes (n = two of 14) or white powder (n = one of 14). Additional evidence for the spread of illicit xylazine comes from five drug seizures made by law enforcement. CONCLUSIONS: Xylazine has penetrated the UK illicit drug market and is not limited to heroin supplies.

Age-specific Reference Intervals of Abbott Intact PTH-Potential Impacts on Clinical Care.

Background: PTH assays are not standardized; therefore, method-specific PTH reference intervals are required for interpretation of results. PTH increases with age in adults but age-related reference intervals for the Abbott intact PTH (iPTH) assay are not available. Methods: Deidentified serum PTH results from September 2015 to November 2022 were retrieved from the laboratory information system of a laboratory serving a cosmopolitan population in central-west England for individuals aged 18 years and older if the estimated glomerular filtration rate was ≥60 mL/min, serum 25-hydroxyvitamin D was >50 nmol/L, and serum albumin-adjusted calcium and serum phosphate were within reference intervals. Age-specific reference intervals for Abbott iPTH were derived by an indirect method using the refineR algorithm. Results: PTH increased with age and correlated with age when controlled for 25-hydroxyvitamin D, estimated glomerular filtration rate, and adjusted calcium (r = 0.093, P < .001). The iPTH age-specific reference intervals for 4 age partitions of 18 to 45 years, 46 to 60 years, 61 to 80 years, and 81 to 95 years were 1.6 to 8.6 pmol/L, 1.8 to 9.5 pmol/L, 2.0 to 11.3 pmol/L, and 2.3 to 12.3 pmol/L, respectively. PTH was higher in women compared with men (P < .001). Sex-specific age-related reference intervals could not be derived because of the limited sample size. Conclusion: Age-specific Abbott iPTH reference intervals were derived. Application of age-specific reference intervals will impact the diagnosis and management of normocalcemic hyperparathyroidism, based on current definitions, and secondary hyperparathyroidism. Additional studies are required to clarify the effect of sex and ethnicity on PTH.

The first drug-related death associated with xylazine use in the UK and Europe.

BACKGROUND AND AIMS: On November 8th, 2022, the United States Food and Drug Administration (FDA) issued a statement alerting healthcare professionals to the increasing prevalence of xylazine in illicit drug overdoses in the country. Xylazine is a veterinary medicine with sedative, analgesic and muscle relaxant properties that is used as a heroin/fentanyl adulterant on the illicit drug market in North America. Here we report the first drug-related death associated with xylazine in the United Kingdom. METHODS: The National Programme on Substance Abuse Deaths (NPSAD) receives reports on drug-related deaths from coroners In England, Wales and Northern Ireland on a voluntary basis. The NPSAD was searched for cases with xylazine detections in cases received by December 31, 2022. RESULTS: One drug-related death associated with xylazine use was reported to NPSAD by December 31, 2022. The deceased was a 43-year-old male who was found dead at home with drug paraphernalia located at the property in May 2022. The post-mortem examination identified recent puncture wounds to the groin. Coronial documentation reports that the deceased had a history of illicit drug use. A number of drugs were detected by post-mortem toxicology and xylazine was implicated in death alongside heroin, fentanyl and cocaine. CONCLUSIONS: To the best of our knowledge, this is the first death associated with xylazine use reported in the UK, and even Europe, and indicates the entry of xylazine into the UK drug supply. This report highlights the importance of monitoring changes in illicit drug markets and the emergence of new drugs.

Outcomes of Transarterial Embolisation (TAE) vs. Transarterial Chemoembolisation (TACE) for Hepatocellular Carcinoma: A Systematic Review and Meta-Analysis.

Although hepatocellular carcinoma is increasingly common, debate exists surrounding the management of patients with unresectable disease comparing transarterial embolisation (TAE) or transarterial chemoembolisation (TACE). This study aimed to compare the outcomes of patients receiving TAE and TACE. A systematic review was performed using PubMed, Medline, Embase, and Cochrane databases to identify randomised controlled trials (RCTs) until August 2021. The primary outcome was overall survival (OS) and the secondary outcomes were progression-free survival (PFS) and adverse events. Five studies with 609 patients were included in the analysis. There was no statistically significant difference in the OS (p = 0.36) and PFS (p = 0.81). There was no difference in OS among patients treated with a single TACE/TAE versus repeat treatments. Post-procedural adverse effects were higher in the TACE group but were not statistically significant. TACE has comparable long-term survival and complications profile to TAE for patients with HCC. However, the low-to-moderate quality of current RCTs warrants high-quality RCTs are necessary to provide enough evidence to give a definitive answer and inform treatment plans for the future.

A systematic review and meta-analysis of miRNAs for the detection of cervical cancer.

Aim: This study aimed to critically appraise the evidence of the diagnostic effectiveness of miRNAs for the detection of cervical cancer. Methods & materials: A systematic review and meta-analysis was performed, searching PubMed, EMBASE and Web of Science. An umbrella meta-analysis of meta-analyses of individual biomarkers was performed. A Grading of Recommendations, Assessment, Development and Evaluations (GRADE) assessment of evidence was also performed. Results: A total of 52 miRNAs were included. Umbrella meta-analysis revealed significant heterogeneity in terms of sensitivity, specificity, receiver operating characteristic (ROC), positive predictive value and/or negative predictive value. Umbrella effects were 0.76 (95% CI: 0.73-0.78), 0.78 (95% CI: 0.75-0.81), 0.77 (95% CI: 0.75-0.80), 0.75 (95% CI: 0.71-0.79) and 0.76 (95% CI: 0.74-0.79), respectively. Conclusion: Moderate quality evidence suggested miR199a-5p, miR21-5p and miR-141a had excellent diagnostic performance.

miRNAs are small chemical messengers that play a role in the regulation of protein produced inside the cytoplasm of cells, including cancer cells. In cervical cancer cells, miRNAs become dysregulated ­ that is, their levels become increased or decreased ­ and therefore detecting their relative abundance or absence in test samples may enable identification of cervical cancer. This study aimed to systematically collect and appraise the evidence for the diagnostic ability of miRNAs for detection of cervical cancer. A systematic appraisal of the evidence was conducted by searching three research databases (PubMed, EMBASE and Web of Science) to collect evidence published up to 13 November 2022. Results for diagnostic performance of 52 miRNAs were extracted from 20 relevant studies. An assessment of risk of bias for each study was performed using a standardized checklist, which identified one high-quality study, 18 moderate-quality studies and one low-quality study. Results for each individual biomarker were assessed by meta-analytic methods, which generated weighted averages for 38 of 52 miRNAs. All 52 miRNAs were then compared using an umbrella meta-analysis (a weighted average of all miRNA biomarkers), which identified significant differences in diagnostic ability between miRNAs. Sensitivity analyses suggested that these differences were partly explained by differences in grades of cervical cancer and differences in types of sample used for testing. A GRADE assessment of the overall evidence quality suggested that moderate-quality evidence supported further investigation of three miRNA biomarkers (miR-199a-5p, miR-21-5p and miR-141a), which performed excellently (i.e., better than the umbrella weighted average) across five performance parameters, including sensitivity, specificity, receiver operator characteristic, positive predictive value and negative predictive value. In summary, this study suggested miR-199a-5p, miR-21-5p and miR-141a had excellent diagnostic performance for detection of cervical cancer and recommends further investigation of these miRNAs in randomized controlled trials.

N-acetylcysteine: a novel approach to methaemoglobinaemia in normothermic liver machine perfusion.

Extended duration of normothermic machine perfusion (NMP) provides opportunities to resuscitate suboptimal donor livers. This intervention requires adequate oxygen delivery typically provided by a blood-based perfusion solution. Methaemoglobin (MetHb) results from the oxidation of iron within haemoglobin and represents a serious problem in perfusions lasting > 24 h. We explored the effects of anti-oxidant, N-acetylcysteine (NAC) on the accumulation of methaemoglobin. NMP was performed on nine human donor livers declined for transplantation: three were perfused without NAC (no-NAC group), and six organs perfused with an initial NAC bolus, followed by continuous infusion (NAC group), with hourly methaemoglobin perfusate measurements. In-vitro experiments examined the impact of NAC (3 mg) on red cells (30 ml) in the absence of liver tissue. The no-NAC group sustained perfusions for an average of 96 (range 87-102) h, universally developing methaemoglobinaemia (≥ 2%) observed after an average of 45 h, with subsequent steep rise. The NAC group was perfused for an average of 148 (range 90-184) h. Only 2 livers developed methaemoglobinaemia (peak MetHb of 6%), with an average onset of 116.5 h. Addition of NAC efficiently limits formation and accumulation of methaemoglobin during NMP, and allows the significant extension of perfusion duration.

A systematic review and meta-analysis of the diagnostic effectiveness of human papillomavirus methylation biomarkers for detection of cervical cancer.

Background: The aim of this systematic review and meta-analysis was to assess the evidence for the diagnostic effectiveness of human papillomavirus (HPV) methylation biomarkers for detection of cervical cancer. Methods: PubMed, Embase and Web of Science were searched. Nine articles focusing on HPV methylation for detection of precancerous and cancerous cervical lesions were included. The QUADAS-2 tool was used for quality assessment. The receiver operating characteristic (ROC) was the main diagnostic performance parameter extracted. Results: Of the nine articles included in this study, seven were of moderate quality and two were of high quality. A meta-analysis of the ROC for 27 HPV methylation biomarkers revealed an overall pooled ROC of 0.770 (95% CI: 0.720-0.819; I2: 98.4%; Q: 1537.4; p < 0.01). Four methylation biomarkers had strong diagnostic ability (ROC > 0.900), 17 were moderate (ROC: 0.7000-0.8999) and six were poor (ROC < 0.700). Conclusion: HPV methylation biomarkers hold significant promise as independent screening tests for the detection of cervical precancerous and cancerous lesions.

This study reviewed the literature to assess the available evidence for the ability of biomarkers based on human papillomavirus (HPV) methylation (i.e., the detection of methyl groups in HPV DNA in cervical specimens) to screen for cervical precancerous and cancerous lesions. Scientific databases were searched, and abstracts screened for relevance. The quality of the included articles was assessed using a quality assessment tool called QUADAS-2. The main diagnostic performance parameter extracted from the included articles was the receiver operating characteristic (ROC), a measure of the ability of a biomarker to detect all true cases (true positives) while excluding all true non-cases (true negatives). After screening, nine articles were included, of which seven were of moderate quality and two were of high quality. ROC data were extracted for 27 biomarkers, of which four methylation biomarkers had high diagnostic ability (i.e., ROC > 0.900), 17 had moderate diagnostic ability (ROC: 0.7000­0.8999) and six had low diagnostic ability (ROC < 0.700). An umbrella meta-analysis (i.e., a weighted-average ROC for all HPV methylation biomarkers) revealed an ROC consistent with moderate diagnostic ability (0.770). The main conclusion from this study was that HPV methylation biomarkers, especially ones with high diagnostic ability, hold significant promise as independent screening tests for the detection of cervical precancerous and cancerous lesions.

Nonadherence in Hypertension: How to Develop and Implement Chemical Adherence Testing.

Nonadherence to antihypertensive medication is common, especially in those with apparent treatment-resistant hypertension (true treatment-resistant hypertension requires exclusion of nonadherence), and its routine detection is supported by clinical guidelines. Chemical adherence testing is a reliable and valid method to detect adherence, yet methods are unstandardized and are not ubiquitous. This article describes the principles of chemical adherence testing for hypertensive patients and provides a set of recommendations for centers wishing to develop the test. We recommend testing should be done in either of two instances: (1) in those who have resistant hypertension or (2) in those on 2 antihypertensives who have a less than 10 mm Hg drop in systolic blood pressure on addition of the second antihypertensive medication. Furthermore, we recommend that verbal consent is secured before undertaking the test, and the results should be discussed with the patient. Based on medications prescribed in United Kingdom, European Union, and United States, we list top 20 to 24 drugs that cover >95% of hypertension prescriptions which may be included in the testing panel. Information required to identify these medications on mass spectrometry platforms is likewise provided. We discuss issues related to ethics, sample collection, transport, stability, urine versus blood samples, qualitative versus quantitative testing, pharmacokinetics, instrumentation, validation, quality assurance, and gaps in knowledge. We consider how to best present, interpret, and discuss chemical adherence test results with the patient. In summary, this guidance should help clinicians and their laboratories in the development of chemical adherence testing of prescribed antihypertensive drugs.

Quantification of Flualprazolam in Blood by LC-MS-MS: A Case Series of Nine Deaths.

The emergence of novel designer benzodiazepines continues to be a public health concern. Flualprazolam is one of these drugs. It was initially identified in 2017. User forums suggest it is slightly more potent than alprazolam and has longer-lasting central nervous system depressant effects. Here we report a simple, sensitive liquid chromatography-tandem mass spectrometry method for flualprazolam and report a series of nine cases in which flualprazolam was quantified. As is typical of forensic toxicology in the twenty-first century, all the cases had more than one drug present. None of the deaths could be directly attributed to flualprazolam alone, but all were likely due to a combination of sedative drugs. However, this paper still adds to the data available to allow interpretation of postmortem flualprazolam concentrations.

Role of liver support systems in the management of post hepatectomy liver failure: A systematic review of the literature.

BACKGROUNDS/AIMS: Post-hepatectomy liver failure (PHLF) is a serious complication following liver resection, with limited treatment options, and is associated with high mortality. There is a need to evaluate the role of systems that support the function of the liver after PHLF. AIMS: The aim of this study was to review the literature and summarize the role of liver support systems (LSS) in the management of PHLF. Publications of interest were identified using systematically designed searches. Following screening, data from the relevant publications was extracted, and pooled where possible. FINDINGS: Systematic review identified nine studies, which used either Plasma Exchange (PE) or Molecular Adsorbent Recirculating System (MARS) as LSS after PHLF. Across all studies, the pooled 90-day mortality rate was 38% (95% CI: 9-70%). However, there was substantial heterogeneity, likely since studies used a variety of definitions for PHLF, and had different selection criteria for patient eligibility for LSS treatment. CONCLUSIONS: The current evidence is insufficient to recommend LSS for the routine management of severe PHLF, with the current literature consisting of only a limited number of studies. There is a definite need for larger, multicenter, prospective studies, evaluating the conventional and newer modalities of support systems, with a view to improve the outcomes in this group of patients.

Nonadherence to antihypertensive medications is related to pill burden in apparent treatment-resistant hypertensive individuals.

OBJECTIVE: Nonadherence to medication is present in at least 50% of patients with apparent treatment-resistant hypertension. We examined the factors associated with nonadherence as detected by a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based urine antihypertensive drug assay. METHODS: All urine antihypertensive test results, carried out for uncontrolled hypertension (BP persistently >140/90 mmHg) between January 2015 and December 2016 at a single toxicology laboratory were analysed. Drugs detected were compared with the antihypertensive drugs prescribed. Patients were classified as adherent (all drugs detected), partially nonadherent (at least one prescribed drug detected) or completely nonadherent (no drugs detected). Demographic and clinical parameters were compared between the adherent and nonadherent groups. Binary logistic regression analysis was performed to determine association between nonadherence and demographic and clinical factors. RESULTS: Data on 300 patients from nine hypertension centres across the United Kingdom were analysed. The median age was 59 years, 47% women, 71% Caucasian, median clinic BP was 176/95 mmHg and the median number of antihypertensive drugs prescribed was four. One hundred and sixty-six (55%) were nonadherent to prescribed medication with 20% of these being completely nonadherent. Nonadherence to antihypertensive medication was independently associated with younger age, female sex, number of antihypertensive drugs prescribed, total number of all medications prescribed (total pill burden) and prescription of a calcium channel blocker. CONCLUSION: This LC-MS/MS urine analysis-based study suggests the majority of patients with apparent treatment-resistant hypertension are nonadherent to prescribed treatment. Factors that are associated with nonadherence, particularly pill burden, should be taken into account while treating these patients.

The Relationship Between Acuity of Organ Failure and Predictive Validity of Sepsis-3 Criteria.

The Sepsis-3 taskforce defined sepsis as suspicion of infection and an acute rise in the Sequential Organ Failure Assessment score by 2 points over the preinfection baseline. Sepsis-3 studies, though, have not distinguished between acute and chronic organ failure, and may not accurately reflect the epidemiology, natural history, or impact of sepsis. Our objective was to determine the extent to which the predictive validity of Sepsis-3 is attributable to chronic rather than acute organ failure. DESIGN: Retrospective cohort study. SETTING: General medicine inpatient service at a tertiary teaching hospital. PATIENTS: A total of 3,755 adult medical acute-care encounters (1,864 confirmed acute infections) over 1 year. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We measured the total Sequential Organ Failure Assessment score at the onset of infection and separated its components (baseline and acute rise) using case-by-case chart reviews. We compared the predictive validities of acuity-focused (acute rise in Sequential Organ Failure Assessment ≥ 2) and conventional (total Sequential Organ Failure Assessment ≥ 2) implementations of Sepsis-3 criteria. Measures of predictive validity were change in the rate of outcomes and change in the area under receiver operating characteristic curves after adding sepsis criteria to multivariate logistic regression models of baseline risk (age, sex, race, and Charlson comorbidity index). Outcomes were inhospital mortality (primary) and ICU transfer or inhospital mortality (secondary). Acuity-focused implementations of Sepsis-3 were associated with neither a change in mortality (2.2% vs 1.2%; p = 0.18) nor a rise in area under receiver operating characteristic curves compared with baseline models (0.67 vs 0.66; p = 0.75). In contrast, conventional implementations were associated with a six-fold change in mortality (2.4% vs 0.4%; p = 0.01) and a rise in area under receiver operating characteristic curves compared with baseline models (0.70 vs 0.66; p = 0.04). Results were similar for the secondary outcome. CONCLUSIONS: The evaluation of the validity of organ dysfunction-based clinical sepsis criteria is prone to bias, because acute organ dysfunction consequent to infection is difficult to separate from preexisting organ failure in large retrospective cohorts.

Oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate-protein interactions.

We describe microarrays of oligosaccharides as neoglycolipids and their robust display on nitrocellulose. The arrays are obtained from glycoproteins, glycolipids, proteoglycans, polysaccharides, whole organs, or from chemically synthesized oligosaccharides. We show that carbohydrate-recognizing proteins single out their ligands not only in arrays of homogeneous oligosaccharides but also in arrays of heterogeneous oligosaccharides. Initial applications have revealed new findings, including: (i) among O-glycans in brain, a relative abundance of the Lewis(x) sequence based on N-acetyllactosamine recognized by anti-L5, and a paucity of the Lewis(x) sequence based on poly-N-acetyllactosamine recognized by anti-SSEA-1; (ii) insights into chondroitin sulfate oligosaccharides recognized by an antiserum and an antibody (CS-56) to chondroitin sulfates; and (iii) binding of the cytokine interferon-gamma (IFN-gamma) and the chemokine RANTES to sulfated sequences such as HNK-1, sulfo-Lewis(x), and sulfo-Lewis(a), in addition to glycosaminoglycans. The approach opens the way for discovering new carbohydrate-recognizing proteins in the proteome and for mapping the repertoire of carbohydrate recognition structures in the glycome.

Validation of a rapid, automated method for the measurement of ethylene glycol in human plasma.

Background Ethylene glycol is a highly toxic compound found in various household products. Cases of poisoning are rare but may be fatal unless diagnosed and treated promptly. Early recognition of poisoning is critical for the management and recovery of patients. Indirect testing is not specific for the presence of ethylene glycol. Therefore, urgent and accurate measurement should be sought if ingestion is suspected in order to determine the need for treatment with an antidote. Here, we present the validation of an automated assay for measurement of ethylene glycol on an Abbott Architect using a commercially available kit (Catachem). Methods Analytical parameters of imprecision, linearity, stability and bias were determined using spiked human plasma samples processed on both the Catachem assay and on an in-house gas chromatography-mass spectrometry method. Interference was assessed using samples collected into a variety of sample collection tubes and spiked with a number of alcohols. Results Excellent agreement was observed between the two methodologies with the enzymatic assay demonstrating linearity and precision across the relevant clinical range (50-3000 mg/L). In addition, the Catachem assay displayed no interference from a number of different sample tubes and alcohols. However, propylene glycol interference was observed at concentrations associated with excessive use (>1 g/L) and 2,3-butanediol interference observed at concentrations associated with butanone ingestion. Inspection of the enzymatic reaction profile was found to differentiate between alcohols. Conclusions This automated assay is suitable for the diagnosis of ethylene glycol poisoning and is now in routine use, enabling the laboratory to provide a rapid 24 h service with support by gas chromatography-mass spectrometry as necessary.

Sequence analysis of alginate-derived oligosaccharides by negative-ion electrospray tandem mass spectrometry.

Negative-ion electrospray tandem mass spectrometry (ES-MS/MS) with collision-induced dissociation (CID) is attempted for sequence determination of alginate oligosaccharides, derived from polyanionic alginic acid, polymannuronate, and polyguluronate by partial depolymerization using either alginate lyase or mild acid hydrolysis. Sixteen homo- and hetero-oligomeric fragments were obtained after fractionation by gel-filtration and strong anion exchange high performance liquid chromatography. The product-ion spectra of these alginate oligosaccharides were dominated by intense B-, C-, Y-, and Z-type ions together with (0,2)A- and (2,5)A-ions of lower intensities. Internal mannuronate residues (M) produce weak but specific decarboxylated Z(int)-ions (Z(int) - 44 Da; int: denotes internal), which can be used for distinction of M and a guluronate residue (G) at an internal position. A reducing terminal M or G, although neither gives rise to a specific ion, can be identified by differences in the intensity ratio of fragment ions of the reducing terminal residue [(2,5)A(red)]/[(0,4)A(red)] (red: denotes reducing terminal).

A Novel 'Dilute-and-Shoot' Liquid Chromatography-Tandem Mass Spectrometry Method for the Screening of Antihypertensive Drugs in Urine.

Arterial hypertension is one of the most preventable causes of premature morbidity and mortality with resistant hypertension reported to be present in 5-30% of the total hypertensive population. Despite the poor prognosis, as many as 53% of those with resistant hypertension are reported to be nonadherent to their prescribed medication. An objective test of adherence, which is easy to administer, quick, inexpensive and reliable, is therefore needed to identify patients with true resistance to antihypertensive drugs to optimize their treatment. We have developed a novel LC-MS-MS method for the detection of 23 commonly prescribed antihypertensive medications in urine. The validated method was subsequently applied to the analysis of urine from a cohort of 49 individuals who were taking at least one antihypertensive agent in the screening profile to determine their adherence. The screening method was found to be reproducible, sensitive and specific with the limit of detection ranging from 0.1 to 1.0 µg/L. Sample preparation is rapid (30 s) and simple, with a total analysis time of 11 min. The assay successfully identified the majority of drugs our cohort had admitted to taking (88%) with drugs not detected in urine, potentially indicating nonadherence to prescribed medication. The performance of this simple, robust LC-MS-MS procedure is suitable for screening urine for the presence of commonly prescribed antihypertensive medications. The assay, which can easily be implemented in other laboratories, has the potential to significantly improve investigation and management of resistant hypertension.

Low maternal vitamin B12 status is associated with lower cord blood HDL cholesterol in white Caucasians living in the UK.

BACKGROUND AND AIMS: Studies in South Asian population show that low maternal vitamin B12 associates with insulin resistance and small for gestational age in the offspring. Low vitamin B12 status is attributed to vegetarianism in these populations. It is not known whether low B12 status is associated with metabolic risk of the offspring in whites, where the childhood metabolic disorders are increasing rapidly. Here, we studied whether maternal B12 levels associate with metabolic risk of the offspring at birth. METHODS: This is a cross-sectional study of 91 mother-infant pairs (n = 182), of white Caucasian origin living in the UK. Blood samples were collected from white pregnant women at delivery and their newborns (cord blood). Serum vitamin B12, folate, homocysteine as well as the relevant metabolic risk factors were measured. RESULTS: The prevalence of low serum vitamin B12 (<191 ng/L) and folate (<4.6 µg/L) were 40% and 11%, respectively. Maternal B12 was inversely associated with offspring's Homeostasis Model Assessment 2-Insulin Resistance (HOMA-IR), triglycerides, homocysteine and positively with HDL-cholesterol after adjusting for age and BMI. In regression analysis, after adjusting for likely confounders, maternal B12 is independently associated with neonatal HDL-cholesterol and homocysteine but not triglycerides or HOMA-IR. CONCLUSIONS: Our study shows that low B12 status is common in white women and is independently associated with adverse cord blood cholesterol.