Timing and dosage of intrapartum prophylactic penicillin for preventing early-onset group B streptococcal disease: assessing maternal and umbilical cord blood concentration.

OBJECTIVE: Timing of administration of antibiotics and concentrations in maternal blood and the umbilical cord blood are important prerequisites for optimal intrapartum antibiotic prophylaxis (IAP) of neonatal early-onset group B streptococcus (GBS) disease. This cohort study aimed to explore penicillin concentrations in mothers and infants at birth in relation to time elapsed from administration to delivery and to the minimal inhibitory concentration (MIC) for GBS. MAIN OUTCOME MEASURES: Penicillin G concentrations in maternal and umbilical cord blood in relation to time and dose from administration to time of delivery. RESULTS: In 44 mother-infant dyads, median maternal penicillin G concentration was 0.2 mg/L (IQR 0-0.8 mg/L; range 0-1.6 mg/L). Median infant penicillin G concentration was 1.2 mg/L (IQR 0.5-5.0 mg/L; range 0-12.7 mg/L). In all infants (N=38) born less than 4 hours after the latest IAP administration, penicillin G concentrations far exceeded MIC (0.125 mg/L), even after short time intervals between IAP administration and birth. The highest plasma concentrations were reached in umbilical cord blood within 1 hour from IAP administration to birth.For 44 mother-infant dyads, maternal concentrations were very low compared with their infants'; particularly, very high concentrations were seen in the 20 infants with only one dose of IAP. CONCLUSION: High concentrations of penicillin G were found in umbilical cord blood of infants born less than 4 hours after IAP administration, well above the MIC for GBS.

Transplacental Transport of Artificial Sweeteners.

The prevalence of obesity is increasing, and the origins of obesity and metabolic dysfunction may be traced back to fetal life. Currently, overweight pregnant women are advised to substitute sugar-sweetened beverages with diet drinks containing artificial sweeteners. Recent evidence suggests that the consumption of artificial sweeteners during pregnancy increases the risk of obesity in the child, but the mechanism is unknown. We hypothesized the transportation of artificial sweeteners across the placenta into the fetal circulation and the amniotic fluid. We included 19 pregnant women who were given an oral dose of acesulfame, cyclamate, saccharin, and sucralose immediately before a planned caesarean section. Nine women were included as controls, and they refrained from an intake of artificial sweeteners. The maternal and fetal blood and amniotic fluid were collected during the caesarean section, and concentrations of artificial sweeteners were measured using mass spectrometry. We found a linear relationship between the fetal plasma concentrations of artificial sweeteners and the maternal plasma concentrations, with adjusted coefficients of 0.49 (95% CI: 0.28-0.70) for acesulfame, 0.72 (95% CI: 0.48-0.95) for cyclamate, 0.51 (95% CI: 0.38-0.67) for saccharin, and 0.44 (95% CI: 0.33-0.55) for sucralose. We found no linear relationship between amniotic fluid and fetal plasma concentrations, but there were positive ratios for all four sweeteners. In conclusion, the four sweeteners investigated all crossed the placenta and were present in the fetal circulation and amniotic fluid.

Reference intervals and stability of haptocorrin and holotranscobalamin in Danish children and elderly.

BACKGROUND: Haptocorrin (HC) and holotranscobalamin (holoTC) carry vitamin B12 (B12) in the circulation and can be useful biomarkers for evaluating B12 status. The concentration of both proteins depends on age, but data on reference intervals for children and the elderly are sparse. Similarly, not much is known about the effect of preanalytical factors. METHODS: HC plasma samples from healthy elderly > 65 years (n = 124) were analysed, and both HC and holoTC were analysed in paediatric serum samples ≤ 18 years (n = 400). Furthermore, we investigated assay precision and stability. RESULTS: HC and holoTC were effected by age. We established reference intervals for HC: 2-10 years, 369-1237 pmol/L; 11-18 years, 314-1128 pmol/L; 65-82 years, 242-680 pmol/L and for holoTC: 2-10 years, 46-206 pmol/L; 11-18 years, 30-178 pmol/L. Analytical coefficients of variations of 6.0-6.8% and 7.9-15.7% were found for HC and holoTC, respectively. HC were affected when stored at room temperature and by freeze/thaw. HoloTC was stable at room temperature and after delayed centrifugation. CONCLUSION: We present novel 95% age-related reference limits for HC and HoloTC in children, and for HC both in children and elderly. Moreover, we found HoloTC to be fairly stable when stored, whereas HC was more vulnerable to preanalytical factors.

Short-term biological variation of plasma uracil in a Caucasian healthy population.

OBJECTIVES: Plasma uracil is a new biomarker to assess the activity of dihydropyrimidine dehydrogenase before cancer treatment with fluoropyrimidine drugs. Knowledge on the biological variation of plasma uracil is important to assess the applicability of plasma uracil as a biomarker of drug tolerance and efficacy. METHODS: A total of 33 apparently healthy individuals were submitted to sequential blood draws for three days. On the second day, blood draws were performed every third hour for 12 h. Plasma uracil was quantified by LC-MS/MS. The within-subject (CVI) and between-subject (CVG) biological variation estimates were calculated using linear mixed-effects models. RESULTS: The overall median value of plasma uracil was 10.6 ng/mL (range 5.6-23.1 ng/mL). The CVI and CVG were 13.5 and 22.1%, respectively. Plasma uracil remained stable during the day, and there was no day-to-day variation observed. No differences in biological variation components were found between sex and no correlation to age was found. Four samples were calculated to be required to estimate the homeostatic set-point ±15% with 95% confidence. CONCLUSIONS: Plasma uracil is subject to tight homeostatic regulation without semidiurnal and day-to-day variation, however between-subject variation exists. This emphasizes plasma uracil as a well-suited biomarker for evaluation of dihydropyrimidine dehydrogenase activity, but four samples are required to establish the homeostatic set-point in a patient.

Automated Interlaboratory Comparison of Therapeutic Drug Monitoring Data and Its Use for Evaluation of Published Therapeutic Reference Ranges.

Therapeutic drug monitoring is a tool for optimising the pharmacological treatment of diseases where the therapeutic effect is difficult to measure or monitor. Therapeutic reference ranges and dose-effect relation are the main requirements for this drug titration tool. Defining and updating therapeutic reference ranges are difficult, and there is no standardised method for the calculation and clinical qualification of these. The study presents a basic model for validating and selecting routine laboratory data. The programmed algorithm was applied on data sets of antidepressants and antipsychotics from three public hospitals in Denmark. Therapeutic analytical ranges were compared with the published therapeutic reference ranges by the Arbeitsgemeinschaft für Neuropsychopharmakologie und Pharmakopsychiatrie (AGNP) and in additional literature. For most of the drugs, the calculated therapeutic analytical ranges showed good concordance between the laboratories and to published therapeutic reference ranges. The exceptions were flupentixol, haloperidol, paroxetine, perphenazine, and venlafaxine + o-desmethyl-venlafaxine (total plasma concentration), where the range was considerably higher for the laboratory data, while the calculated range of desipramine, sertraline, ziprasidone, and zuclopenthixol was considerably lower. In most cases, we identified additional literature supporting our data, highlighting the need of a critical re-examination of current therapeutic reference ranges in Denmark. An automated approach can aid in the evaluation of current and future therapeutic reference ranges by providing additional information based on big data from multiple laboratories.

Plasma concentration of orally administered amoxicillin and clindamycin in patients receiving haemodialysis.

OBJECTIVES: In the randomized controlled trial PANTHEM, the prophylactic effect of oral amoxicillin or clindamycin is investigated in patients receiving chronic haemodialysis (HD). However, data on plasma concentrations of these antibiotics during HD are sparse. This study aims to determine if the plasma concentration of amoxicillin and clindamycin is sufficient during HD after oral administration of amoxicillin and clindamycin at three different time intervals prior to the HD procedure. METHODS: Adult patients receiving chronic HD were investigated twice with an interval of at least 7 days starting with either a tablet of 500/125 mg amoxicillin/clavulanic acid or a tablet of 600 mg clindamycin. Patients were randomized to take the antibiotics either 30, 60 or 120 min prior to the HD procedure. Plasma antibiotic concentrations were measured at start, midway and at the end of HD. A lower threshold was set at 2.0 mg/L for amoxicillin and at 1.0 mg/L for clindamycin. In addition, a population pharmacokinetic (PK) analysis was performed, assessing PTA. RESULTS: In the amoxicillin cohort (n = 37), 84% of patients and 95% of all plasma amoxicillin concentrations were above or at the threshold throughout the dialysis procedure. In the clindamycin cohort (n = 33), all concentrations were above the threshold throughout the dialysis procedure. Further, in all patients, the mean plasma concentration of both amoxicillin and clindamycin across the HD period was well above the threshold. Finally, the PK model predicted a high PTA in the majority of patients. DISCUSSION: In patients on chronic HD, oral administration of amoxicillin/clavulanic acid (500/125 mg) or clindamycin (600 mg) within 30-120 min prior to HD leads to a sufficient prophylactic plasma concentration across the HD period.

Instability of uracil in whole blood might affect cancer treatment with fluoropyrimidines.

BACKGROUND AND AIMS: Measurement of plasma uracil is used before cancer treatment with fluoropyrimidines to determine if patients tolerate a full dose. Incorrect preanalytical handling may cause falsely elevated concentration and result in suboptimal cancer treatment. We aimed to examine the stability of uracil in whole blood stored at room temperature (RT) and the effect of centrifugation temperature. MATERIALS AND METHODS: EDTA tubes (6x4 mL) were collected from 25 healthy volunteers. Five samples were stored 0, 1.5, 2, 3, and 4 h at RT and centrifuged at 4 °C. The sixth sample was centrifuged at RT after 1.5 h. Uracil was measured using an in-house LC-MS/MS method. RESULTS: Storage of whole blood at RT followed by centrifugation at 4 °C caused a rapid increase in uracil concentration. Already after 1.5 h, the mean change (20.5 % (95 % CI: 11.9-29.2 %)) exceeded the maximum permissible difference. Centrifugation at RT instead of 4 °C after 1.5 h resulted in a smaller increase (7.0 % (95 % CI: 0.7-13.4 %)), although not statistically significant (p = 0.0527). CONCLUSION: Uracil was unstable in samples processed according to current recommendations. Our data indicates better stability when centrifugation is performed at RT compared with 4 °C but further research into this is necessary.

Artificial Sweeteners in Breast Milk: A Clinical Investigation with a Kinetic Perspective.

Artificial sweeteners (ASs) are calorie-free chemical substances used instead of sugar to sweeten foods and drinks. Pregnant women with obesity or diabetes are often recommended to substitute sugary products with ASs to prevent an increase in body weight. However, some recent controversy surrounding ASs relates to concerns about the risk of obesity caused by a variety of metabolic changes, both in the mother and the offspring. This study addressed these concerns and investigated the biodistribution of ASs in plasma and breast milk of lactating women to clarify whether ASs can transfer from mother to offspring through breast milk. We recruited 49 lactating women who were provided with a beverage containing four different ASs (acesulfame-potassium, saccharin, cyclamate, and sucralose). Blood and breast milk samples were collected before and up to six hours after consumption. The women were categorized: BMI < 25 (n = 20), BMI > 27 (n = 21) and type 1 diabetes (n = 8). We found that all four ASs were present in maternal plasma and breast milk. The time-to-peak was 30−120 min in plasma and 240−300 min in breast milk. Area under the curve (AUC) ratios in breast milk were 88.9% for acesulfame-potassium, 38.9% for saccharin, and 1.9% for cyclamate. We observed no differences in ASs distributions between the groups.

New methods for quantification of amoxicillin and clindamycin in human plasma using HPLC with UV detection.

OBJECTIVES: We aimed to develop simple and rapid HPLC methods for determination of amoxicillin and clindamycin in human plasma. METHODS: Plasma samples were pretreated by direct deproteinization with acetonitrile and the analytical separation took place on a reverse phase Poroshell 120 EC-C18 column (2.7 µm, 2.1 × 100 mm) with a gradient of acetonitrile. UV detection at 229 nm for amoxicillin and 204 nm for clindamycin was used for determination of the antibiotics in plasma. RESULTS: The calibration curves were linear over the concentration ranges of 1-100 mg/L for amoxicillin and 1-15 mg/L for clindamycin with a correlation coefficient of ≥0.98. Intra-assay precisions were all ≤15% and the accuracies were within ±15%. The limit of quantification (LOQ) was found to be 0.5 mg/L for amoxicillin and 1 mg/L for clindamycin with inter-assay imprecision coefficient of variances (CVs) of 18.7% and 15.6%, respectively. The present HPLC methods were successfully applied on spike-in samples and on plasma samples collected 4-6 and 3.5-5.5 h after oral antibiotic administration of 500 mg of amoxicillin and 600 mg of clindamycin, respectively. CONCLUSIONS: We have developed HPLC methods with UV detection for quantification of amoxicillin and clindamycin in human plasma. The methods are fast, simple and suitable for use in routine settings and clinical studies.

Development and validation of an LC-MS/MS method for the quantification of artificial sweeteners in human matrices.

Artificial sweeteners are widely used as substitutes for sugar. The sweeteners are generally considered safe, however their whereabouts during pregnancy and lactation and the effect on child development are poorly explored. There is a need for new tools to measure these substances during pregnancy and lactation. Here, we describe the development and validation of a sensitive liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of acesulfame, cyclamate, saccharin and sucralose in human plasma, umbilical cord blood, amniotic fluid and breast milk. The samples were prepared by protein precipitation and separated on a Luna Omega Polar C18 column (2.1 × 50 mm, 1.6 µm). Electrospray ionization in negative mode and multiple reaction monitoring were used to monitor the ion transitions. The validated concentration ranges were from 1 to 500 ng/ml (10-500 ng/ml for sucralose). Interassay precisions were all ≤15% and the accuracies were within ±15%. Stability, linearity, dilution integrity, carryover and recovery were also examined and satisfied the validation criteria. Finally, this analytical method was successfully applied on spiked samples of plasma, umbilical cord blood, amniotic fluid and breast milk, proving its suitability for use in clinical studies on artificial sweeteners, including during pregnancy and lactation.

Delayed sample arrival at the laboratory does not lead to more false negatives in the Danish population screening for colorectal cancer.

In Denmark, biennial population screening for colorectal cancer was introduced in 2014 for all aged 50-74 years. Five laboratories representative for the regional division of Denmark perform the immunochemical testing of faecal occult blood in the screening samples (iFOBT, OC-Sensor (Eiken Chemical, cut-off 100 µg/L)). In July 2016, a new agreement on the public post-delivery entailed an increased lag time (five days) from the screening participant drops the screening sample into a mail-box until sample arrival at the laboratories. Previous work had reported that a lag time above five days led to more false negative iFOBT tests. We investigated if this was true also under Danish conditions. We performed two stability tests; one with sample storage at 30 °C for 14 days (N = 60), and another with sample storage at room temperature for 13 days (N = 10). We extracted data from our laboratory information system (LABKA) on all iFOBT tests performed in the entire Central Denmark Region (N = 104,328 patients) during the last six months for each calendar year 2014-16. For each year, we computed the distribution of iFOBT tests below and above cut-off. Our stability tests showed no positive samples switching to false negative after storage; however, some negative samples turned false positive, especially at 30 °C. The data showed no change in the distribution of iFOBT tests below and above cut-off after July 2016. We found no evidence that an enhanced lag time increased the number of false negative iFOBT tests in the Danish screening program for colorectal cancer.

Three family members with elevated plasma cobalamin, transcobalamin and soluble transcobalamin receptor (sCD320).

BACKGROUND: Plasma cobalamin is requested in order to diagnose cobalamin deficiency and low levels confirm a deficient state. Here, we present three family members with unexpected high levels of cobalamin. METHODS: We included a patient referred for cobalamin measurement due to neurological symptoms, her son and her daughter. Mother and son both suffered from myotonic dystrophy type II, while the daughter tested negative for this disease. Blood samples were analyzed for cobalamin, haptocorrin, transcobalamin, holoTC, and sCD320. We employed gel filtration and antibody precipitation for further characterization. The protein coding region of the TCN2 gene, encoding transcobalamin, was sequenced. RESULTS: The patient, her {son} and [daughter] all had cobalamin levels above the measurement range of the routine method employed (>1476 pmol/L). Total transcobalamin and (holoTC) were 5980 (1500), {5260 (2410)} and [5630 (1340)] pmol/L, which is well above the upper reference limits of 1500 (160) pmol/L. The sCD320 concentration was also well above the upper reference limit of 97 arb.u.: 1340, {1510} and [1090] arb.u. Haptocorrin levels were within the reference range and no signs of cobalamin deficiency were found. DNA sequencing of the TCN2 gene revealed several known polymorphisms not associated with highly elevated transcobalamin levels. Upon gel filtration, sCD320 eluted as a larger molecule than previously reported. By incubation with anti-transcobalamin antibodies, we precipitated both transcobalamin and part of sCD320. CONCLUSIONS: The high cobalamin levels were mainly explained by high levels of holoTC, possibly caused by complex formation with its soluble receptor, sCD320. The family occurrence points to a genetic explanation.

Holotranscobalamin, a marker of vitamin B-12 status: analytical aspects and clinical utility.

Approximately one-quarter of circulating cobalamin (vitamin B-12) binds to transcobalamin (holoTC) and is thereby available for the cells of the body. For this reason, holoTC is also referred to as active vitamin B-12. HoloTC was suggested as an optimal marker of early vitamin B-12 deficiency >20 y ago. This suggestion led to the development of suitable assays for measurement of the compound and clinical studies that aimed to show the benefit of measurement of holoTC rather than of vitamin B-12. Today holoTC can be analyzed by 3 methods: direct measurement of the complex between transcobalamin and vitamin B-12, measurement of vitamin B-12 attached to transcobalamin, or measurement of the amount of transcobalamin saturated with vitamin B-12. These 3 methods give similar results, but direct measurement of holoTC complex is preferable in the clinical setting from a practical point of view. HoloTC measurement has proven useful for the identification of the few patients who suffer from transcobalamin deficiency. In addition, holoTC is part of the CobaSorb test and therefore useful for assessment of vitamin B-12 absorption. Clinical studies that compare the ability of holoTC and vitamin B-12 to identify individuals with vitamin B-12 deficiency (elevated concentration of methylmalonic acid) suggest that holoTC performs better than total vitamin B-12. To date, holoTC has not been used for population-based assessments of vitamin B-12 status, but we suggest that holoTC is a better marker than total vitamin B-12 for such studies.

Transcobalamin deficiency caused by compound heterozygosity for two novel mutations in the TCN2 gene: a study of two affected siblings, their brother, and their parents.

Transcobalamin (TC) deficiency (OMIM# 275350) is a rare, autosomal recessive disorder that presents in early infancy with a broad spectrum of symptoms, including failure to thrive, megaloblastic anemia, immunological deficiency, and neurological symptoms. Here we report a study of a family (parents and three children) with two children suffering from TC deficiency caused by two different mutations in the TCN2 gene. Initially, molecular genetic analysis of genomic DNA revealed a heterozygous mutation in the +1 position of exon 7 (c.1106+1 G > A) in the father and all three children. Bioinformatic analysis indicates that this mutation causes exon skipping, and further experiments supported this hypothesis and suggested that the mutant allele undergoes nonsense-mediated messenger RNA (mRNA) decay. We did not identify further mutations in genomic DNA that could explain TC deficiency in the two children. However, further efforts using complementary DNA (cDNA) derived from RNA from blood leukocytes identified a large deletion removing the entire exon 8, resulting in a frameshift and a premature stop codon (p.E371fsX372) in the mother and the two affected children. Our data indicate that if exon-by-exon DNA sequencing of genomic DNA does not uncover mutations corresponding to the phenotype, a systematic search for other mutations should be initiated by sequencing cDNA or using semiquantitative methods to detect large deletions in TCN2.