Assessing the prevalence of alcohol consumption in early pregnancy using blood biomarker analysis: a consistent pattern across north-east England?

BACKGROUND: We previously investigated the prevalence of alcohol consumption in early pregnancy in Northumbria Healthcare NHS Foundation Trust, a locality of north-east England. The prevalence was 1.4% based on blood sample biomarker analysis using carbohydrate deficient transferrin (CDT) and 3.5% for gamma-glutamyltransferase (GGT). AIMS: To supplement this research by investigating the prevalence of alcohol use using identical methods in a different locality of the same region. METHODS: Six-hundred random blood samples taken at the antenatal booking appointment were anonymously analysed for the presence of CDT, a validated marker of chronic alcohol exposure (normalizing 2-3 weeks from abstinence) and GGT, a liver enzyme elevated for up to 8 weeks after alcohol exposure. RESULTS: The North Tees and Hartlepool NHS Foundation Trust data revealed a CDT prevalence rate of 1.7% (95% CI: 0.7-2.9) and GGT prevalence rate of 4.2% (95% CI: 2.6-5.9). However, these measures are not sensitive to low levels of alcohol; and no overlapping cases were identified or a significant correlation demonstrated between CDT or GGT. DISCUSSION: These data support our earlier work. Prevalence rates according to CDT and GGT analysis were similar in both areas, suggesting similar patterns of sustained alcohol use in pregnancy across the region.

Assessing prevalence of alcohol consumption in early pregnancy: Self-report compared to blood biomarker analysis.

Providing appropriate antenatal and postnatal care for women who drink alcohol in pregnancy is only possible if those at risk can be identified. We aimed to compare the prevalence of alcohol consumption in the first trimester of pregnancy using self-report and blood biomarker analysis. Six-hundred routine blood samples from 2014, taken at the antenatal booking appointment, in the first trimester of pregnancy, were anonymously analysed for the presence of Carbohydrate Deficient Transferrin (CDT), a validated marker of chronic alcohol exposure (normalising 2-3 weeks from abstinence) and Gamma-glutamyltransferase (GGT), a liver enzyme elevated for up to 8 weeks after alcohol exposure. In a separate sample of women, from 2015, data taken during the antenatal visit, documenting women's self-reported alcohol consumption, were collected. The percentage of women who reported alcohol intake in the first trimester was 0.8%. This compared to 74.1% of women who reported consuming alcohol before pregnancy. CDT analysis revealed a prevalence rate of 1.4% and GGT a prevalence rate of 3.5% in the first trimester of pregnancy. Although those with elevated CDT generally had high levels of GGT, only one person was positive for CDT and GGT. Results from CDT analysis and self-report may underestimate prevalence for different reasons. GGT appeared to lack specificity, but it may have value in supporting findings from CDT analysis. Further studies using additional blood biomarkers, or a combination of blood biomarkers and self-report, may be beneficial in accurately detecting alcohol drinking history in pregnancy.

How strong is the evidence for using blood biomarkers alone to screen for alcohol consumption during pregnancy? A systematic review.

Accurate and early identification of women at risk from alcohol consumption during pregnancy allows education and support programmes to be targeted at those most in need. We aimed to conduct a systematic review to compare the efficacy of blood analysis and maternal self-report in detecting at risk women during pregnancy. This review investigated diagnostic accuracy. We searched four databases (Medline, Embase, Psychinfo and CINAHL) for relevant articles and conducted hand searches of recent issues of key journals in the field. No restriction was placed on inclusion in terms of publication date or language. Studies were deemed eligible if they were original research and included a direct comparison of the results of blood biomarker analysis and self-reported alcohol use for the detection of alcohol consumption in pregnant women. Quality appraisal of included studies was conducted using the QUADAS II tool. Eight studies met the inclusion criteria. Gamma-glutamyltransferase (GGT) was investigated in five studies, mean corpuscular volume (MCV) and phosphatidylethanol (PEth) in three studies and carbohydrate deficient transferrin (CDT), aspartate aminotransferase (AST), alanine aminotransferase (ALT) and whole blood associated acetaldehyde assay (WBAA) were each investigated in two studies. Although all of the studies were rated of good methodological quality, none of the biomarkers had both high sensitivity and specificity when compared to self-report. There was some evidence that a combination of biomarkers, or combining biomarkers with self-report, increases accuracy. In summary, the blood biomarkers examined were of limited use in screening for low and moderate alcohol consumption in pregnancy when compared to self-report. However, certain biomarkers, such and CDT and PEth may complement self-report and help improve the accuracy of diagnosis.

Concentration Monitoring of Plasma Ribavirin: Validation of a Liquid Chromatography-Mass Spectrometric Method and Clinical Sample Collection.

BACKGROUND: A liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for routine measurement of ribavirin concentrations in EDTA-anticoagulated plasma. METHODS: After protein precipitation, we used a bridged ethylene hybrid (hydrophilic interaction) chromatography column, 0.1 mmol/L ammonium formate pH 3.0, and a gradient of 85%-96% acetonitrile to achieve baseline separation of ribavirin from isobaric uridine. Quantitation was assured using both primary (m/z 245.3 > 113.0) and secondary transitions (m/z 245.3 > 96.0) of the protonated species. Chromatographic separation and column washing also negated interference from major phospholipid species. RESULTS: There was a linear relationship between concentration and response to 10 mg/L, with a minimum detectable level and a minimum level of quantitation both of 0.1 mg/L. Imprecision within the assay was <10% at 0.1 mg/L and <6% between assays for concentrations >0.4 mg/L. Bias was <4%. In clinical samples (n = 12), there was no difference in ribavirin concentrations obtained by an established liquid chromatographic assay with ultraviolet detection. Ribavirin concentrations were stable in plasma stored at room temperature for 3 days but then decreased significantly on day 7. Plasma concentrations were stable for 15 weeks at -20 °C. Concentrations in plasma separated from whole blood at room temperature fell by a median of 19.4% at 4 hours and then rose substantially (median 251% by 3 days). Dose-normalized ribavirin concentrations reached a steady state after a mean of >6 weeks treatment in 76 patients with hepatitis C. CONCLUSIONS: A hydrophilic interaction liquid chromatography-tandem mass spectrometric method to measure ribavirin in plasma was developed. Samples for ribavirin estimation should be kept at 4 °C, separated within 2 hours of collection and stored at 4 °C before analysis, with long-term storage at -20 °C. This method was applied to a study of the ribavirin therapeutic monitoring in patients with hepatitis C.

A direct method for the measurement of everolimus and sirolimus in whole blood by LC-MS/MS using an isotopic everolimus internal standard.

BACKGROUND: Whole-blood concentrations of the immunosuppressant drugs everolimus and sirolimus should be monitored. A sensitive and selective method offering the detection of both analytes in small sample volumes would optimize the throughput of samples for sirolimus or everolimus analysis. This study reports the validation of a liquid chromatography tandem mass spectrometry method, including a stable isotope internal standard, for the simultaneous measurement of everolimus and sirolimus. METHODS: Whole-blood samples (20 µL) were treated with ammonium bicarbonate (20 µL), zinc sulfate (20 µL), and internal standard solution ((13)C(2)D(4)-everolimus in acetonitrile, 100 µL). After centrifugation, 20 µL of the supernatant was injected onto a Waters Symmetry C18 high-performance liquid chromatography column. The aqueous and organic mobile phases were 2 mmole/L of ammonium acetate containing 0.1% (vol/vol) formic acid, in water and methanol, respectively. Analytes were detected using tandem mass spectrometry (Waters Acquity TQD). RESULTS: Analytes were eluted at around 2 minutes within a 6-minute analytical run time. Detector response was linear for both analytes across the ranges studied (1-49 µg/L for sirolimus, 1-41 µg/L for everolimus), and a lower limit of quantitation of 1 µg/L was reliably attained. Intraassay and interassay imprecision and inaccuracy were <15% (coefficient of variation) in all cases. Analyte recovery was in the range of 72%-117%. The analytes were stable in blood after freezing and thawing, and for at least 12 hours after preparation while waiting to be injected. Ion suppression and interference from phospholipids were not significant. CONCLUSIONS: A straightforward, robust liquid chromatography tandem mass spectrometry assay has been developed and validated for the simultaneous measurement of everolimus and sirolimus in small volumes of whole blood.

Mycophenolate monitoring in liver, thoracic, pancreas, and small bowel transplantation: a consensus report.

Assessing the value of mycophenolic acid (MPA) monitoring outside renal transplantation is hindered by the absence of any trial comparing fixed-dose and concentration-controlled therapy. However, in liver and thoracic transplantation particularly, clinical trials, observational studies with comparison groups, and case series have described MPA efficacy, exposure/efficacy relationships, pharmacokinetic variability, and clinical outcomes relating to plasma MPA concentrations. On the basis of this evidence, this report identifies MPA as an immunosuppressant for which the combination of variable disposition, efficacy, and adverse effects contributes to interindividual differences seemingly in excess of those optimal for a fixed-dosage mycophenolate regimen. Combined with experiences of MPA monitoring in other transplant indications, the data have been rationalized to define circumstances in which measurement of MPA concentrations can contribute to improved management of mycophenolate therapy in nonrenal transplant recipients.

An investigation into the bias between liquid chromatography-tandem mass spectrometry and an enzyme multiplied immunoassay technique for the measurement of mycophenolic acid.

Mycophenolic acid is now the second most widely used immunosuppressant in solid organ transplantation. Overestimation of mycophenolic acid concentration is a recognized problem of immunoassay, and high-performance liquid chromatography with ultraviolet detection methods have long analysis times and a risk of analyte coelution which may compromise high sample throughput in a clinically meaningful time frame. A novel liquid chromatography-tandem mass spectrometry assay for mycophenolic acid was developed using very small (10 microL) sample volumes and evaluated in comparison with an established immunological assay. The enzyme mediated immunoassay showed a median positive bias compared with liquid chromatography-tandem mass spectrometry of 14.6%. Linear regression analysis showed a significant positive impact of bilirubin (r2 = 0.230) on bias with further increases of r2 to 0.261, 0.286, and 0.294 with the stepwise addition of creatinine, hematocrit, and gamma-glutamyl transpeptidase, respectively. The impact of comedication and transplant type depended on the patient population: analysis of all samples showed opposing effects to analysis of those samples lacking data with biochemical variables above. The liquid chromatography-tandem mass spectrometry method described in this report is capable of measuring mycophenolic acid concentrations in very small sample volumes and in a timely fashion without the significant overestimates characterizing enzyme mediated immunoassay measurements in patients with serologic features characterizing liver or renal graft rejection.

Calcineurin inhibitor sparing in paediatric solid organ transplantation : managing the efficacy/toxicity conundrum.

Despite their efficacy, the calcineurin inhibitors (CNIs) ciclosporin and tacrolimus carry a risk of debilitating adverse effects, especially nephrotoxicity, that affect the long-term outcome and survival of children who are given organ transplants. Simple reduction in dosage of CNI has little or no long-term benefit on their adverse effects, and complete withdrawal without threatening graft outcome may only be possible after liver transplantation. Until the last decade, the only option was to increase corticosteroid and/or azathioprine doses, which imposed additional long-term hazards. Considered here are the emerging generation of new agents offering an opportunity for improving long-term graft survival, minimizing CNI-related adverse events and ensuring patient well-being.A holistic, multifaceted strategy may need to be considered - initial selection and optimized use and monitoring of immunosuppressant regimens, early recognition of indicators of patient and graft dysfunction, and, where applicable, early introduction of CNI-sparing regimens facilitating CNI withdrawal. The evidence reviewed here supports these approaches but remains far from definitive in paediatric solid organ transplantation. Because de novo immunosuppression uses CNI in more than 93% of patients, reduction of CNI-related adverse effects has focused on CNI sparing or withdrawal.A recurring theme where sirolimus and mycophenolate mofetil have been used for this purpose is the importance of their early introduction to limit CNI damage and provide long-term benefit: for example, long-term renal function critically reflects that at 1 year post-transplant. While mycophenolic acid shows advantages over sirolimus in preserving renal function because the latter is associated with proteinuria, sirolimus appears the more potent immunosuppressant but also impairs early wound healing. The use of CNI-free immunosuppressant regimens with depleting or non-depleting antibodies plus sirolimus and mycophenolic acid needs much wider investigation to achieve acceptable rejection rates and conserve renal function. The adverse effects of the alternative immunosuppressants, particularly the dyslipidaemia associated with sirolimus, needs to be minimized to avoid replacing one set of adverse effects (from CNIs) with another. While we can only conjecture that judicious combinations with the second generation of novel immunosuppressants currently in development will provide these solutions, a rationale of low-dose therapy with multiple immunosuppressants acting by complementary mechanisms seems to hold the promise for efficacy with minimal toxicity until the vision of tolerance achieves reality.

Immunosuppression in pediatric solid organ transplantation: opportunities, risks, and management.

The pediatric transplant community stands at a time of unprecedented choice of immunosuppressive agents - and with a legacy of morbidity from those agents used in the previous two decades. This review considers the clinical utility and side-effect profiles of immunosuppressants used widely in current practice (e.g., glucocorticoids, azathioprine, ciclosporin, tacrolimus, mycophenolate, and sirolimus) and those agents which are in increasing use or in evaluation (e.g., IL-2 receptor antibodies, everolimus, FTY720, LEA29Y, and deoxyspergualin). Further consideration is given to the wider drug interactions likely during the use of new immunosuppressant regimens and to our growing awareness of the influences of genetic heterogeneity on drug efficacy and handling. Finally, we consider the new demands being placed on the use of drug monitoring to regulate dosage of this new repertoire of immunosuppressants.

Low hematocrit and serum albumin concentrations underlie the overestimation of tacrolimus concentrations by microparticle enzyme immunoassay versus liquid chromatography-tandem mass spectrometry.

BACKGROUND: Rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods are used increasingly for tacrolimus (TRL) monitoring but show a negative difference with respect to a microparticle immunoassay (MEIA). This report examines possible reasons for this difference between methods. METHODS: We collected 1156 blood samples from 277 adult and 121 pediatric recipients of liver, renal, and bone marrow grafts or hepatocyte or pancreatic islet cell implants. TRL was measured in whole blood by MEIA and LC-MS/MS, and hematologic and biochemical data were collected when available. RESULTS: LC-MS/MS was significantly more precise (P <0.02) than the MEIA with increased sensitivity. The MEIA had a median difference of 16.2% vs LC-MS/MS overall, and this was significantly affected by patient cohort (P <0.001). The difference was greater in adult or pediatric liver graft recipients while they were inpatients rather than outpatients (31.8% and 14.0% vs 7.5% and 6.5%, respectively). The difference was also greater in bone marrow than kidney graft recipients (32.8% vs 15.8%, respectively). Multiple linear regression analysis showed significant inverse relationships of this difference with hematocrit (packed cell volume) and plasma albumin (P <0.001) in the total cohort and a positive relationship with plasma bilirubin in a subgroup of pediatric liver graft recipients. CONCLUSIONS: Patients with a low packed cell volume and plasma albumin are likely to show artificially high concentrations of TRL when measured by MEIA. The increased risk of underimmunosuppression must be considered should doses be reduced to lower these seemingly high TRL concentrations.

Mycophenolic acid pharmacokinetics in pediatric liver transplant recipients.

The aim of this study is to study mycophenolic acid (MPA) pharmacokinetics in stable pediatric liver transplant recipients and determine which times best represent the area under the concentration versus time curve (AUC) of MPA plasma concentrations. MPA pharmacokinetic profiles were determined in 21 liver transplant recipients (age, 2 to 15 years; 12 boys) administered mycophenolate mofetil (MMF) for at least 6 months. Ten patients were coadministered cyclosporine A (CsA), and 11 patients were coadministered tacrolimus (Tac). Plasma MPA levels were analyzed by enzyme-multiplied immunoassay technique in blood samples at 0, 0.33, 0.67, 1.25, 2, 3.5, 5, and 7 hours after MMF administration. The AUC of plasma concentrations to 7 hours (AUC(0-7)) was calculated using the linear trapezoidal rule. MPA plasma trough concentration (C(0)), maximal concentration, and AUC(0-7) values ranged 9- to 14-fold at a median of 1.81 mg/L (range, 0.4 to 3.7 mg/L), 10.5 mg/L (range, 2.8 to 40.0 mg/L), and 30.2 mg/L.hr (range, 9.3 to 80.3 mg/L.hr), respectively. AUC(0-7) correlated significantly with MMF dose (r = 0.552; P =.010) and C(0) (r = 0.844; P <.001). Median AUC(0-7) (29.6 v 31.4 mg/L.hr; P =.918) was similar in children comedicated with CsA or Tac. Median MMF dose was greater in the CsA group (500 v 250 mg; P =.006). Consequently, median AUC(0-7) was significantly lower in the CsA group when equalized for dose and body weight (2.02 v 3.85 microg/L.hr per mg of MMF dose per kg of weight; P =.002). Variations of MPA pharmacokinetics in pediatric liver transplant recipients suggest that monitoring MPA plasma levels is required. C(0) correlates closely with AUC. Comedication with CsA increased MMF dosage requirements compared with children on Tac therapy.

Evidence for metabolic and endocrine abnormalities in subjects recovered from anorexia nervosa.

Subjects with anorexia nervosa (AN) at low weight display metabolic, endocrine, and behavioral abnormalities. Whether these various differences are a consequence of the condition and persist after recovery is unclear. We tested the hypothesis that abnormalities in the insulin and leptin axes and in the desire to eat persisted in subjects who had recovered from AN in terms of body mass index (BMI) and menstrual function. Endocrine, metabolic, and psychological parameters were assessed by sampling under fasting conditions and serially in response to a standard meal. Subjects included 18 females recovered from AN and 18 female controls and measures included plasma insulin, leptin, glucose and beta-hydroxybutyrate (beta-HBA) concentrations together with desire to eat. Fasting glucose concentrations were normal in both groups, but fasting insulin concentrations were significantly lower and the fasting glucose/insulin ratio significantly higher in the recovered subjects. The glucose concentration was significantly higher at the end of the meal period in the recovered group. The peak increase of insulin during the meal was significantly less in the recovered group and in response to the meal, glucose/insulin ratios were significantly higher for the first 45 minutes indicating a delayed insulin response. Fasting beta-HBA concentrations were not significantly different between groups, but postmeal decreases were significant and larger in the recovered AN group. Fasting and meal-related leptin concentrations were not significantly different between the groups and in both groups were correlated with BMI. In controls, but not in recovered subjects, the reported desire to eat was correlated with plasma glucose and leptin concentrations. The insulin, glucose and beta-HBA data indicated the presence of insulin hypersensitivity in the recovered subjects. As the insulin response to the meal was blunted and apparently delayed, there may be a persistent alteration in pancreatic function as a long-term pathological consequence of the anorexia. Alternatively, these data indicate a possible trait marker for AN.

Mycophenolic acid and mycophenolic acid glucuronide pharmacokinetics in pediatric liver transplant recipients: effect of cyclosporine and tacrolimus comedication.

Determinants of the wide interindividual variability of the pharmacokinetics of mycophenolic acid (MPA) in 21 stable pediatric liver transplant recipients were investigated in relation to the kinetics of the drug's major phenolic glucuronide metabolite (MPAG), cyclosporin (CsA), or tacrolimus (Tac) co-medication and liver and renal function. Trough concentrations (C(0) ) most reliably predicted the area under the curve (AUC) of 0-7 hours MPA plasma concentrations (r (2) = 0.650). Co-medication with CsA demanded higher MPA mofetil (MMF) doses to achieve equivalent trough levels than Tac (362 vs. 178 mg per mg/L, P= 0.004). Median MPA C(0) (range) was significantly lower during CsA co-therapy when corrected for MMF dose (2.8 vs. 5.6 mg MPA/L for Tac, P= 0.006). The AUC of MPAG was correspondingly higher during CsA co-medication (229 vs. 94 mg/L/h for Tac, P = 0.012) with the MPA-to-MPAG ratio at C(0) correspondingly lower (0.10 vs. 0.14, respectively, P = 0.04). This suggested contrasting effects of CsA and Tac on MPA glucuronidation or its excretion and enterohepatic recirculation. MPAG AUC was correlated to body weight and creatinine clearance. Children with elevated aspartate transaminase (AST; but with no evidence of rejection on liver biopsy, n = 7) had significantly lower MPA trough levels compared with those in whom AST was normal (0. 77 vs. 1.76 mg/L, P = 0.05), but there was no difference in the MMF dose per body weight. Examination of the MPA profiles in these subjects showed significantly lower MPA concentrations from 120 minutes after dose until the end of the 7-hour profile and suggest an accelerated clearance or decreased enterohepatic recirculation.)