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

Nanoscale interaction layer at the interface between Al films and SiO2 substrates of Al/AlOx/Al Josephson tunnel junctions

An interaction layer is found at the Al/SiO2 interface in Al/AlOx/Al tunnel junctions grown on SiO2 substrates. The amorphous intermixing layer has an average thickness of about 5 nm. We present the detailed structure of this interfacial layer as determined by transmission electron microscopy. The layer contains alumina with aluminum being octahedrally coordinated according to electron energy loss spectroscopy analysis rather than tetrahedrally coordinated, where the latter coordination is the most common type in amorphous alumina. Depth profiles of the Al-O and Si-O bonding characteristics were also investigated using energy loss near edge structure

Direct observation of the thickness distribution of ultra thin AlOx barriers in Al/AlOx/Al Josephson junctions

We have directly measured the thickness distribution of the tunnel barriers in state-of-the-art Al/AlOx/Al tunnel junctions. From the distribution we can conclude that less than 10% of the junction area dominates the electron tunnelling. The barriers have been studied by transmission electron microscopy, specifically using atomic resolution annular dark field (ADF) scanning transmission electron microscopy (STEM) imaging. The direct observation of the local barrier thickness shows a Gaussian distribution of the barrier thickness variation along the junction, from ~1 to ~2nm. We have investigated how the thickness distribution varies with oxygen pressure (Po) and oxidation time (to) and we find, in agreement with resistance measurements, that an increased to has a larger impact on barrier thickness and its uniformity compared to an increased Po

Micronutrient Status and Other Correlates of Hemoglobin among Children with Stunting : A Cross-Sectional Study in Uganda

In low-income countries, undernutrition and infections play a major role in childhood anemia. Stunted children may be at particular risk of anemia. In a cross-sectional study nested in a nutrition trial among 12–59-month-old stunted children in eastern Uganda, we measured hemoglobin (Hb) and markers of iron, cobalamin, folate and vitamin A status. We assessed low micronutrient status, socio-demography, stunting severity, inflammation and malaria as correlates of Hb and anemia using linear and logistic regression analyses, respectively. Of 750 stunted children, the mean ± SD age was 32.0 ± 11.7 months and 55% (n = 412) were male. The mean Hb was 104 ± 15 g/L and 65% had anemia, Hb 8.3 mg/L (−6.2 g/L, 95% CI: −8.4; −4.0), plasma folate <20 nmol/L (−4.6 g/L, 95% CI: −8.1;−1.1), cobalamin < 222 pmol/L (−3.0 g/L, 95% CI: −5.4; −0.7) and serum retinol-binding protein < 0.7 µmol/L (−2.0 g/L, 95% CI: −4.1; 0.2). In addition, severe stunting, inflammation and malaria were negative correlates. Anemia is common among stunted children in eastern Uganda; micronutrient deficiencies, inflammation and malaria are associated with low Hb.Peer reviewe

Correlates of Iron, Cobalamin, Folate, and Vitamin A Status among Stunted Children : A Cross-Sectional Study in Uganda

Micronutrient deficiencies and stunting are prevalent. We assessed correlates of iron, cobalamin, folate, and vitamin A biomarkers in a cross-sectional study of stunted children aged 12–59 months in eastern Uganda. The biomarkers measured were serum ferritin (S-FE), soluble transferrin receptor (S-TfR), retinol binding protein (S-RBP), plasma cobalamin (P-Cob), methylmalonic acid (P-MMA), and folate (P-Fol). Using linear regression, we assessed socio-demography, stunting severity, malaria rapid test, and inflammation as correlates of micronutrient biomarkers. Of the 750 children, the mean (SD) age was 32.0 (11.7) months, and 45% were girls. Iron stores were depleted (inflammation-corrected S-FE 8.3 mg/L). P-Cob was low (0.75 µmol/L). Inflammation-corrected S-RBP was low (<0.7 µmol/L) in 21% and P-Fol (<14 nmol/L) in 1%. Age 24–59 months was associated with higher S-FE and P-Fol and lower S-TfR. Breastfeeding beyond infancy was associated with lower iron status and cobalamin status, and malaria was associated with lower cobalamin status and tissue iron deficiency (higher S-TfR) despite iron sequestration in stores (higher S-FE). In conclusion, stunted children have iron, cobalamin, and vitamin A deficiencies. Interventions addressing stunting should target co-existing micronutrient deficiencies.Peer reviewe

Effect of lipid-based nutrient supplements on micronutrient status and hemoglobin among children with stunting: secondary analysis of a randomized controlled trial in Uganda.

BACKGROUND: Micronutrient deficiencies and anemia are widespread among children with stunting. OBJECTIVES: We assessed the effects of lipid-based nutrient supplements (LNS) containing milk protein (MP) and/or whey permeate (WP) on micronutrient status and hemoglobin (Hb) among children with stunting. METHODS: This was a secondary analysis of a randomized controlled trial. Children aged 12-59 mo with stunting were randomly assigned to LNS (100 g/d) with milk or soy protein and WP or maltodextrin for 12 wk, or no supplement. Hb, serum ferritin (S-FE), serum soluble transferrin receptor (S-TfR), plasma cobalamin (P-Cob), plasma methylmalonic acid (P-MMA), plasma folate (P-Fol), and serum retinol-binding protein (S-RBP) were measured at inclusion and at 12 wk. Data were analyzed using linear and logistic mixed-effects models. RESULTS: Among 750 children, with mean age ± SD of 32 ± 11.7 mo, 45% (n = 338) were female and 98% (n = 736) completed follow-up. LNS, compared with no supplementation, resulted in 43% [95% confidence interval (CI): 28, 60] greater increase in S-FE corrected for inflammation (S-FEci), 2.4 (95% CI: 1.2, 3.5) mg/L greater decline in S-TfR, 138 (95% CI: 111, 164) pmol/L greater increase in P-Cob, 33% (95% CI: 27, 39) reduction in P-MMA, and 8.5 (95% CI: 6.6, 10.3) nmol/L greater increase in P-Fol. There was no effect of LNS on S-RBP. Lactation modified the effect of LNS on markers of cobalamin status, reflecting improved status among nonbreastfed and no effects among breastfed children. LNS increased Hb by 3.8 (95% CI: 1.7, 6.0) g/L and reduced the odds of anemia by 55% (odds ratio: 0.45, 95% CI: 0.29, 0.70). MP compared with soy protein increased S-FEci by 14% (95% CI: 3, 26). CONCLUSIONS: LNS supplementation increases Hb and improves iron, cobalamin, and folate status, but not vitamin A status among children with stunting. LNS should be considered for children with stunting. This trial was registered at ISRCTN as 13093195

The Cobalamin-Binding Protein in Zebrafish Is an Intermediate between the Three Cobalamin-Binding Proteins in Human

In humans, three soluble extracellular cobalamin-binding proteins; transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are involved in the uptake and transport of cobalamin. In this study, we investigate a cobalamin-binding protein from zebrafish (Danio rerio) and summarize current knowledge concerning the phylogenetic evolution of kindred proteins. We identified a cobalamin binding capacity in zebrafish protein extracts (8.2 pmol/fish) and ambient water (13.5 pmol/fish) associated with a single protein. The protein showed resistance toward degradation by trypsin and chymotrypsin (like human IF, but unlike human HC and TC). The cobalamin analogue, cobinamide, bound weaker to the zebrafish cobalamin binder than to human HC, but stronger than to human TC and IF. Affinity for another analogue, adenosyl-pseudo-cobalamin was low compared with human HC and TC, but high compared with human IF. The absorbance spectrum of the purified protein in complex with hydroxo-cobalamin resembled those of human HC and IF, but not TC. We searched available databases to further explore the phylogenies of the three cobalamin-binding proteins in higher vertebrates. Apparently, TC-like proteins are the oldest evolutionary derivatives followed by IF and HC (the latter being present only in reptiles and most but not all mammals). Our findings suggest that the only cobalamin-binding protein in zebrafish is an intermediate between the three human cobalamin binders. These findings support the hypothesis about a common ancestral gene for all cobalamin-binding proteins in higher vertebrates

Mouse Transcobalamin Has Features Resembling both Human Transcobalamin and Haptocorrin

In humans, the cobalamin (Cbl) -binding protein transcobalamin (TC) transports Cbl from the intestine and into all the cells of the body, whereas the glycoprotein haptocorrin (HC), which is present in both blood and exocrine secretions, is able to bind also corrinoids other than Cbl. The aim of this study is to explore the expression of the Cbl-binding protein HC as well as TC in mice. BLAST analysis showed no homologous gene coding for HC in mice. Submaxillary glands and serum displayed one protein capable of binding Cbl. This Cbl-binding protein was purified from 300 submaxillary glands by affinity chromatography. Subsequent sequencing identified the protein as TC. Further characterization in terms of glycosylation status and binding specificity to the Cbl-analogue cobinamide revealed that mouse TC does not bind Concanavalin A sepharose (like human TC), but is capable of binding cobinamide (like human HC). Antibodies raised against mouse TC identified the protein in secretory cells of the submaxillary gland and in the ducts of the mammary gland, i.e. at locations where HC is also found in humans. Analysis of the TC-mRNA level showed a high TC transcript level in these glands and also in the kidney. By precipitation to insolubilised antibodies against mouse TC, we also showed that >97% of the Cbl-binding capacity and >98% of the Cbl were precipitated in serum. This indicates that TC is the only Cbl-binding protein in the mouse circulation. Our data show that TC but not HC is present in the mouse. Mouse TC is observed in tissues where humans express TC and/or HC. Mouse TC has features in common with both human TC and HC. Our results suggest that the Cbl-binding proteins present in the circulation and exocrine glands may vary amongst species

Metformin Lowers Serum Cobalamin without Changing Other Markers of Cobalamin Status: A Study on Women with Polycystic Ovary Syndrome

Treatment with the anti-diabetic drug metformin is followed by a decline in plasma cobalamin, but it is unsettled whether this denotes an impaired cobalamin status. This study has explored changes in the markers of cobalamin status in women with Polycystic Ovary Syndrome treated with metformin (1.5–2.5 g per day) (n = 29) or placebo (n = 23) for six months. Serum samples were collected before and after two, four, and six months of treatment. We found serum cobalamin to decline and reach significant lower levels after six months of treatment (p = 0.003). Despite the decline in serum cobalamin, we observed no reductions in the physiological active part of cobalamin bound to transcobalamin (holotranscobalamin), or increase in the metabolic marker of cobalamin status, methylmalonic acid. Instead, the non-functional part of circulating cobalamin bound to haptocorrin declined (p = 0.0009). Our results have two implications: The data questions whether metformin treatment induces an impaired cobalamin status in PCOS patients, and further suggests that serum cobalamin is a futile marker for judging cobalamin status in metformin-treated patients

Development of a Sensitive ELISA for Gastric Intrinsic Factor and Detection of Intrinsic Factor Immunoreactivity in Human Serum

Gastric Intrinsic Factor (IF) is produced by the parietal cells of the stomach and secreted into the gastrointestinal tract where it ensures the active absorption of vitamin B12. We hypothesized that a small amount of IF ends up in the circulation and can be measured in serum. The aim of this study was to develop an assay for measuring human IF and to demonstrate its presence in serum. We designed a sensitive ELISA for measurement of human IF using a commercial monoclonal antibody and an in-house polyclonal antibody as capture and detecting antibody, respectively. Imprecision, accuracy, and linearity of the assay were examined. We established a reference interval based on serum samples from 240 healthy donors, and explored the daily IF fluctuations in 20 healthy subjects. Employing a prototype IF ELISA and size exclusion chromatography experiments, we demonstrated the presence of IF in human serum. In its final design, the IF ELISA has a measurement range of 0.2 to 50 pmol/L. The intra-assay and total imprecision were 7.9% and 15%, respectively. The 95% reference interval (18&ndash;65 years) was 1.7&ndash;11.6 pmol/L. No diurnal fluctuation or notable sex differences were observed. Our results suggest that the assay is capable of detecting and quantifying human IF in the circulation and may prove useful in the characterization of patients with impaired IF production