Metabolism of 25-Hydroxy-Vitamin D in Human Macrophages Is Highly Dependent on Macrophage Polarization.

Macrophages synthesize active vitamin D (1,25-dihydroxy-vitamin D) and express the vitamin D receptor in the nucleus; however, vitamin D metabolism in relation to macrophage polarization and function is not well understood. We studied monocyte-derived macrophages (MDMs) from human buffy coats polarized into M0, M1 (LPS + IFNγ), M2a (IL4 + IL13) and M2c (IL10) macrophage subtypes stimulated with 25-hydroxy-vitamin D (1000 and 10,000 nanomolar). We measured vitamin D metabolites (25-hydroxy-vitamin D, 1,25-dihydroxy-vitamin D, 24,25-dihydroxy-vitamin D and 3-epi-25-hydroxy-vitamin D) in cell media with liquid chromatography-mass spectrometry-mass spectrometry. The mRNA expression (CYP27B1, CYP24A1 and CYP24A1-SV) was measured with qPCR. We found that reparative MDMs (M2a) had significantly more 1,25-dihydroxy-vitamin D compared to the other MDMs (M0, M1 and M2c). All MDMs were able to produce 3-epi-25-hydroxy-vitamin D, but this pathway was almost completely attenuated in inflammatory M1 MDMs. All MDM subtypes degraded vitamin D through the 24-hydroxylase pathway, although M1 MDMs mainly expressed an inactive splice variant of CYP24A1, coding the degrading enzyme. In conclusion, this study shows that vitamin D metabolism is highly dependent on macrophage polarization and that the C3-epimerase pathway for vitamin D is active in macrophages.

Validating registry data on statins prescriptions by blood measurements.

PURPOSE: To validate prescription registry data as a measurement of adherence to statins through a direct method using assays for selected statins in serial blood samples collected from two prospective cohorts of Danish colorectal cancer patients. METHODS: We linked information on statin prescriptions from the Aarhus University Prescription Database with the cancer cohorts from Aalborg University Hospital. For statin-prescribed patients, we calculated a prescription window covering the anticipated duration of the prescription. For each statin-prescribed patient with at least one blood sample in a prescription window, we selected without replacement a never-statin-prescribed patient matched on sex, age, and calendar year of surgery. Each of the selected blood samples were analyzed using assays to detect statins. We calculated the positive and negative predictive value of the prescription registry reporting using the assay result as the gold standard. RESULTS: We identified 73 ever-statin-prescribed patients with a total of 253 blood samples and 74 blood samples among never-statin-prescribed patients. The positive predictive value for prescribed patients, with presence of statins in at least one blood sample as the gold standard, was 93% (95% CI, 86%-97%) and the negative predictive value was 93% (95% CI, 86%-97%). Stratified results did not reveal substantial differences in predictive values. Fifty-two (71%) of the statin-prescribed patients had statins in every blood sample, suggesting continuous adherence. CONCLUSION: This study showed a high adherence with treatment with statins among colorectal cancer patients.

Unmeasurable low vitamin D levels caused by a novel, homozygote loss-of-function variant in the group-specific component gene.

A 29-year-old female, born to consanguineous parents, was found with unmeasurable levels of vitamin D (<10 nmol/L) after routine biochemical screening during her first pregnancy. She did not respond to either oral or intramuscular vitamin D supplementation and was an otherwise healthy young woman, with no signs of rickets, osteomalacia, osteoporosis, or secondary hyperparathyroidism. Western blot analysis revealed total lack of vitamin D binding protein, and next generation sequencing confirmed a novel, pathogenic homozygote loss-of-function mutation in exon 13 of the group-specific component gene, that encodes the poly A tail for vitamin D binding protein. She was therefore diagnosed with hereditary DBP deficiency, and vitamin D supplementation was diminished to life-long regular vitamin D supplementation (25 µg per day). This case is extremely interesting, as it expands our knowledge of vitamin D physiology and supports the free hormone hypothesis, given that the patient was asymptomatic despite no measurable levels of vitamin D.

Analytical validation of the Roche 25-OH Vitamin D Total assay.

BACKGROUND: Vitamin D deficiency is considered a major health issue and therefore there is a need for reliable routine tests for measurement of the vitamin in blood samples. Here we present a validation of the recently released Roche 25-OH Vitamin D Total assay (Vitamin D Total). METHODS: We analyzed control materials (2 levels) and patient serum pools (3 levels) ranging from 34 to 123 nmol/L 84 times over a period of 21 days, and we analyzed five serum pools in 10 separate runs to verify the limit of quantification. We also analyzed 53 paired samples of serum and Li-heparin plasma. We evaluated the 25-OH Vitamin D Total assay in comparison to our in-house liquid chromatography tandem mass spectrometry (LC-MS/MS) method [194 patient samples without 25-hydroxy vitamin D(2) (25OHD(2)) and 23 patient samples containing 25OHD(2)]. RESULTS: At concentrations of 34 and 56 nmol/L within-run CVs were 4.8% and 1.9% and total CVs were 8.3% and 6.1%. We verified that the limit of quantification was 22.5 nmol/L, as stated by the manufacturer. No significant difference was observed between serum and plasma samples (Li-heparin). Comparison with LC-MS/MS using 194 samples containing 25OHD(3) only (no 25OHD(2)) showed Vitamin D Total nmol/L=1.07×(LC-MS/MS) nmol/L+4.7 nmol/L, whereas comparison of 25OHD(2) using 23 patient samples showed Vitamin D Total nmol/L=0.55×(LC-MS/MS) nmol/L-2.38 nmol/L (Demings regression). CONCLUSIONS: The Roche Vitamin D Total assay is judged suitable for measurement of 25OHD in serum and Li-heparin plasma. Results for 25OHD(3) are comparable to those obtained by LC-MS/MS, while results for 25OHD(2) are around half of those obtained by LC-MS/MS.

Local concentrations of gentamicin obtained by microdialysis after a controlled application of a GentaColl sponge in a porcine model.

Local treatment with gentamicin may be an important tool in the prevention and treatment of surgical site infections in high-risk procedures and patients. The aim of this study was to evaluate the pharmacokinetic profile of gentamicin in bone and surrounding tissue, released from a controlled application of a GentaColl sponge in a porcine model. In eight female pigs, a GentaColl sponge of 10 × 10 cm (1.3 mg gentamicin/cm2 ) was placed in a cancellous bone cavity in the proximal tibia. Microdialysis was used for sampling of gentamicin concentrations over 48 hours from the cavity with the implanted GentaColl sponge, cancellous bone parallel to the cavity over and under the epiphyseal plate, cortical bone, the intramedullary canal, subcutaneous tissue, and the joint cavity of the knee. Venous blood samples were obtained as reference. The main finding was a mean peak drug concentration (95% CI) of gentamicin in the cancellous bone cavity containing the implanted GentaColl sponge of 11 315 (9049-13 581) µg/mL, persisting above 1000 µg/mL until approximately 40 hours after application. Moreover, the concentrations were low (<1 µg/mL) in the surrounding tissues as well as in plasma. The mean peak gentamicin concentration from the cancellous bone cavity after a controlled application of a GentaColl sponge was high and may be adequate for the prevention of biofilm formation. However, high MIC strains and uncontrolled application of the GentaColl sponge may jeopardize this conclusion.

Correlations between calcineurin phosphatase inhibition and cyclosporine metabolites concentrations in kidney transplant recipients: implications for immunoassays.

Cyclosporine exhibits a wide spectrum of metabolites that vary considerably in the extent to which they interfere with the various parent drug monitoring immunoassays. There is no consensus regarding the clinical significance of metabolites. Cyclosporine exerts its immunosuppressive action by inhibiting the enzyme calcineurin phosphatase. Determination of the enzyme's activity is one of the most promising pharmacodynamic markers. It is unknown how calcineurin phosphatase inhibition correlates with various cyclosporine monitoring assays and what is the potential impact of metabolites in this perspective? The aim of the present study was to determine the concentration of cyclosporine (by means of three different assay methods) and the four most significant metabolites (AM1, AM4N, AM9, and AM1C) in relation to calcineurin phosphatase inhibition. Twelve randomly selected cyclosporine-treated renal transplant patients were included in the study. Blood samples were drawn before, 1, 2, 3, 4, 6, 8, and 12 hr after oral intake of cyclosporine. Parent drug and metabolites were determined by liquid chromatography/tandem mass spectrometry (LC/MSMS). Additionally, cyclosporine concentration was determined by the enzyme multiplied immunoassay technique (EMIT) and by the polyclonal fluorescence polarization immunoassay (pFPIA). Calcineurin phosphatase activity was measured by its ability to dephosphorylate a previously phosphorylated 19-amino acid peptide. We found that calcineurin phosphatase inhibition correlates strongly with parent cyclosporine metabolites concentrations determined by all three assay methods. Determination methods that took metabolites into consideration exhibit stronger correlations with calcineurin phosphatase inhibition (sum of cyclosporin plus metabolites r=-0.93, LC/MSMS; pFPIA r=-0.94, P

Vitamin D content in human breast milk: a 9-mo follow-up study.

BACKGROUND: Parents are advised to avoid the direct sun exposure of their newborns. Therefore, the vitamin D status of exclusively breastfed newborns is entirely dependent on the supply of vitamin D from breast milk. OBJECTIVES: We explored concentrations of ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3) (vitamin D) and 25-hydroxivitamin D2 plus D3 (25-hydroxyvitamin D [25(OH)D]) in foremilk and hindmilk during the first 9 mo of lactation and identified indexes of importance to the concentrations. DESIGN: We collected blood and breast-milk samples from mothers at 2 wk (n = 107), 4 mo, (n = 90), and 9 mo (n = 48) postpartum. Blood samples from infants were collected 4 and 9 mo after birth. We measured concentrations of vitamin D metabolites in blood and milk samples with the use of liquid chromatography-tandem mass spectrometry. RESULTS: Concentrations of vitamin D and 25(OH)D correlated significantly and were higher in hindmilk than in foremilk. Milk concentrations were also correlated with maternal plasma 25(OH)D concentrations. In foremilk and hindmilk, concentrations were a median (IQR) of 1.35% (1.04-1.84%) and 2.10% (1.63-2.65%), respectively, of maternal plasma 25(OH)D concentrations (P < 0.01). Milk concentrations showed a significant seasonal variation. Mothers who were taking vitamin D supplements had higher concentrations than did nonusers. Medians (IQRs) of infant daily intake through breast milk of vitamin D and 25(OH)D were 0.10 µg (0.02-0.40 µg) and 0.34 µg (0.24-0.47 µg), respectively, which were equal to a median (IQR) antirachitic activity of 77 IU/d (52-110 IU/d). CONCLUSIONS: The supply of vitamin D from breast milk is limited. Exclusively breastfed infants received <20% of the daily dose recommended by the Institute of Medicine for infants during the first year of life. This trial was registered at clinicaltrials.gov as NCT02548520.

Vitamin D depletion does not affect key aspects of the preeclamptic phenotype in a transgenic rodent model for preeclampsia.

Maternal vitamin D deficiency is proposed as a risk factor for preeclampsia in humans. We tested the hypothesis that vitamin D depletion aggravates and high supplementation ameliorates the preeclampsia phenotype in an established transgenic rat model of human renin-angiotensin system-mediated preeclampsia. Adult rat dams, transgenic for human angiotensinogen (hAGT) and mated with male rats transgenic for human renin (hREN), were fed either vitamin D-depleted chow (VDd) or enriched chow (VDh) 2 weeks before mating and during pregnancy. Mean blood pressure was recorded by tail-cuff, and 24-hour urine samples were collected in metabolic cages at days 6 and 18 of gestation. Rats were sacrificed at day 21 of gestation. Depleted dams (VDd) had negligible serum 25-hydroxyvitamin D2+3 levels (mean ± SEM; 2.95 ± 0.45 nmol/l vs. VDh 26.20 ± 2.88 nmol/l, P = .01), but in both groups, levels of 1,25(OH)2D3 remained below detection level of 25 pmol/l. Dietary vitamin D depletion did not aggravate hypertension (mean ± SEM BP, day 20 of gestation: 151.38 ± 5.65 mmHg VDd vs. 152.00 ± 4.10 mmHg VDh) or proteinuria. Fetal anthropometrics were similar between the groups, whereas VDd displayed lower placental:fetal weight ratios (0.15 vs. 0.16 g/g, P = .01) and increased sFlt-1/PlGF ratio. Expression of hREN was lower in placenta of VDd dams (0.82 ± 0.44 AU vs. 1.52 ± 0.15 AU, P = .04). Expression of key vitamin D metabolizing enzymes was unchanged. Dietary vitamin D intervention did not alter key aspects of the preeclampsia phenotype using the transgenic rodent model of human renin-angiotensin system-mediated pre-eclampsia, plausibly due to altered vitamin D metabolism or excretion in the transgenic rats.

Development of a high-resolution melting genotyping assay for the angiotensin I converting enzyme insertion/deletion polymorphism and establishment of genotype-specific reference intervals in a Danish population.

BACKGROUND: The serum-angiotensin I converting enzyme (s-ACE) activity is influenced by a genetic insertion/deletion (I/D) polymorphism in the ACE gene, and the resulting large interindividual variation in s-ACE limits the use of normal reference intervals in the evaluation of sarcoidosis. In this study, we developed a new method for genotyping the I/D polymorphism in ACE and established genotype-specific reference intervals in order to improve the diagnostic accuracy and the value for treatment of sarcoidosis. METHODS: The new genotyping assay is based on high-resolution melting (HRM) using LCGreen + and was used to genotype 400 healthy Danish individuals. The assay was compared to a real-time polymerase chain reaction (RT-PCR) assay in a validation set of 86 samples. Enzyme activity in serum was measured using the Infinity™ ACE Liquid Stable Reagent from Thermo adapted for the ABX Pentra analyzer. RESULTS: There was full concordance between genotyping assays. The three genotypes II, ID and DD were present with a frequency of 0.23, 0.51 and 0.26. The distribution of s-ACE values in the total population was non-Gaussian (non-parametric 95% reference interval 12.0-60.0 U/L). The median activities of the genotypes differed significantly (P<0.0001). Ninety-five per cent non-parametric reference intervals for the subpopulations were determined to 6.3-38.5, 14.0-56.0 and 23.3-71.2 U/L for II, ID and DD, respectively. CONCLUSION: We have developed a simple and robust method for ACE genotyping and determined genotype-specific reference intervals for s-ACE concentrations in the Danish population. The new reference intervals may increase the value of s-ACE measurements.

High-throughput liquid-liquid extraction and LCMSMS assay for determination of circulating 25(OH) vitamin D3 and D2 in the routine clinical laboratory.

BACKGROUND: Increasing focus on vitamin D as essential to health has underscored the need for accurate and precise high-throughput measurement of serum 25(OH)D. METHODS: Serum was denatured in acetonitrile containing hexadeuterated 25(OH)D3 as internal standard (IS) and automatically applied to filter plates packed with inert diatomous earth material for subsequent heptane extraction. Extracts were chromatographed on a C12 HPLC column, and detected on a triple quadropole mass spectrometer. RESULTS: The inter-assay precision was 9.4% and 8.8% respectively at 32.0 and 59.7 nmol/l for 25(OH)D3 and 8.6% and 8.0% at 23.4 and 64.4 nmol/l for 25(OH)D2. The detection limit was 10 nmol/l for both metabolites. Three percent of samples contained >50 nmol/l 25(OH)D2. Total run time was 4 min. We have performed more than 200,000 routine samples and the method performs well in external control schemes. CONCLUSION: We describe a robust, high-throughput, LLE-LCMSMS method for accurate and precise quantitation of 25(OH)D3 and 25(OH)D2 in serum. The use of diatomaceous earth material for extraction of vitamin D in 96-well format enables fast, simple and efficient sample preparation. The method offers a cost-effective alternative to immunological methods for use in the routine clinical biochemical laboratory.

The calcineurin activity profiles of cyclosporin and tacrolimus are different in stable renal transplant patients.

Cyclosporin and tacrolimus remain the cornerstone immunosuppressive drugs in organ transplantation. Dosing and monitoring these drugs is based on pharmacokinetic protocols, but measuring a pharmacodynamic parameter, calcineurin phosphatase (CaN) activity, could be a valuable supplement in determining optimal doses. Forty stable renal transplant patients were investigated three times in a 6-month period. Blood samples were drawn at 0, 1, 2, 3 and 4 h after oral intake of tacrolimus (FK) or cyclosporin at days 1 and 180. At day 90, one blood sample at trough level (FK) or C2 level (cyclosporin A, CsA) was drawn. CaN activity was determined in whole blood as the release of 32P from a phosphorylated peptide. Activity of the 32P was quantitated by liquid scintillation and results converted to Units CaN, utilizing a calibration curve with CaN. We demonstrated that calcineurin activity profiles at days 1 and 180 were the same for both drugs. Furthermore, we found that patients treated with tacrolimus or cyclosporin displayed different calcineurin activity profiles. We found that cyclosporin displayed greater calcineurin inhibition than tacrolimus. We have demonstrated that the two drugs exert significantly different effects on calcineurin activity in renal transplant patients with stable, well-functioning grafts and that tacrolimus-treated patients can maintain good, stable graft function with minimal CaN inhibition.

Role of metabolites and calcineurin inhibition on C2 monitoring in renal transplant patients.

BACKGROUND: Many transplantation centres have switched to C2 monitoring of cyclosporin-treated renal transplant patients. The rationale is that the C2 correlates best with AUC0-4 (area under the concentration-time curve), which again correlates with rejection and nephrotoxicity. It has also been demonstrated that calcineurin phosphatase is inhibited maximally 1-2 h after intake of cyclosporin in patients receiving their first dose. Cyclosporin is metabolized to many compounds, which may influence the results of immunoassays. Some metabolites may have immunosuppressive activity. METHODS: Cyclosporin metabolites were added to whole blood from healthy volunteers and the calcineurin phosphatase activity (CaN) was determined. Twenty renal transplant patients at varying times after transplantation had blood samples drawn in the morning before and 1, 2, 3 and 4 h after intake of their usual dose of cyclosporin microemulsion. Whole blood samples were analysed by liquid chromatography/tandem mass spectrometry for cyclosporin blood concentration and for the cyclosporin metabolites AM1, AM9, AM1c and AM4n. All samples were analysed for CaN utilizing a 32P-labelled 19 amino-acid peptide. RESULTS: The concentrations of AM1c and AM4n were very low and cannot contribute to CaN inhibition. The ratio of AM1 and AM9 to cyclosporin was high before intake of the drug, but it was much lower during the following 4 h. The 2-h values of cyclosporin were the best predictors of AUC0-4. Calcineurin phosphatase was most inhibited in the 2-h samples and the 2-h value of CaN was the best predictor of CaN AUC0-4. The correlation with calcineurin inhibition seemed better for cyclosporin plus metabolites than for cyclosporin. CONCLUSIONS: Samples collected at 2 h are the best predictors of AUC0-4 for both cyclosporin and calcineurin inhibition. The impact of metabolites appears to be small; however, the temporal profile of calcineurin inhibition seemed to follow cyclosporin plus metabolites better than cyclosporin alone.

Liquid chromatography tandem mass spectrometry assay for topiramate analysis in plasma and cerebrospinal fluid: validation and comparison with fluorescence-polarization immunoassay.

SUMMARY: The authors report the development and validation of a liquid chromatography tandem mass spectrometry assay (LC/MS/MS assay) for the analysis of topiramate (2,3:4,5-bis-o-(-1-methyl)-beta-D-fructopyranose sulfamate) in plasma and cerebrospinal fluid (CSF). Comparison is made with the commercially available fluorescence-polarization immunoassay (FPIA). LC/MS/MS ASSAY: Using the internal standard, 1,2:3,4-bis-o-(1-methylethylidene-alpha-D-galactopyranose sulfamate), a structural isomer, the calibration curve in plasma was linear in the concentration range of 0.02-20.0 mg/L (r(2) = 0.9998). The coefficients of variation in plasma were < or = 3%, and the accuracy ranged from 100% to 101% in the therapeutically relevant concentration range of 0.4-16.0 mg/L. In CSF, the mean recovery was 98%, and there was linearity between the nominal and the estimated concentration in the range of 1.5-20.0 mg/L (r(2)= 0.9996). FPIA: The calibration curve was linear in the concentration interval of 1.6-24.3 mg/L (r(2) = 0.9994), and the mean recovery was 96%. Accuracy in plasma was 99- 104%, and precision was 3.2-6.0%. In CSF, there was linearity between the nominal concentration and the estimated concentration in the range of 1.5-20.0 mg/L (r(2) = 0.9995), and the mean recovery was 100%. COMPARISON BETWEEN FPIA AND LC/MS/MS: There was a high correlation between the FPIA and the LC/MS/MS assay (r(2) = 0.9965 in plasma and r(2) = 0.9996 in CSF, P < 0.001 for both). In plasma and CSF, the two methods showed equal results, evaluated as the ratio between the two methods (plasma: median ratio = 1.00; 95% confidence interval [CI], 0.98-1.02, paired-sample test, P = 0.79; and CSF: median ratio = 1.00, 95% CI, 0.99-1.02, paired-sample test, P = 0.75). The coefficient of variation on the ratios between the two methods had similar levels: 5% in plasma and 3% in CSF. CONCLUSION: The new LC/MS/MS assay has favorable characteristics, being highly precise and accurate. FPIA also proved precise and accurate, and there was a high agreement with the LC/MS/MS assay in plasma and CSF. Either method displayed sufficient precision and accuracy and may thus be implemented in daily routine.