Corrigendum: Effects of Vitamin D and K on Interleukin-6 in COVID-19.

[This corrects the article DOI: 10.3389/fnut.2021.761191.].

Effects of Vitamin D and K on Interleukin-6 in COVID-19.

BACKGROUND: Pathology during COVID-19 infection arises partly from an excessive inflammatory response with a key role for interleukin (IL)-6. Both vitamin D and K have been proposed as potential modulators of this process. METHODS: We assessed vitamin D and K status by measuring circulating 25-hydroxyvitamin D (25(OH)D) and desphospho-uncarboxylated Matrix Gla-Protein (dp-ucMGP), respectively in 135 hospitalized COVID-19 patients in relation to inflammatory response, elastic fiber degradation and clinical outcomes. RESULTS: Comparing good and poor disease outcomes of COVID-19 patients, vitamin 25(OH)D levels were not significantly different. IL-6 levels, however, were significantly higher in patients with poor outcome, compared to patients with good outcome (30.3 vs. 153.0 pg/mL; p < 0.0001). Dp-ucMGP levels as biomarker of extrahepatic vitamin K status was associated with IL-6 levels (r = 0.35; p < 0.0001). In contrast, 25(OH)D levels were only borderline statistically significant correlated with IL-6 (r = -0.14; p <0.050). A significant association was also found between IL-6 and elastic fiber degradation. Contrary to vitamin K status, 25(OH)D did not correlate with elastic fiber degradation. CONCLUSIONS: Dp-ucMGP associates with IL-6 as a central component of the destructive inflammatory processes in COVID-19. An intervention trial may provide insight whether vitamin K administration, either or not in combination with vitamin D, improves clinical outcome of COVID-19.

Reduced Vitamin K Status as a Potentially Modifiable Risk Factor of Severe Coronavirus Disease 2019.

BACKGROUND: Respiratory failure and thromboembolism are frequent in severe acute respiratory syndrome coronavirus 2-infected patients. Vitamin K activates both hepatic coagulation factors and extrahepatic endothelial anticoagulant protein S, required for thrombosis prevention. In times of vitamin K insufficiency, hepatic procoagulant factors are preferentially activated over extrahepatic proteins. Vitamin K also activates matrix Gla protein (MGP), which protects against pulmonary and vascular elastic fiber damage. We hypothesized that vitamin K may be implicated in coronavirus disease 2019 (COVID-19), linking pulmonary and thromboembolic disease. METHODS: A total of 135 hospitalized COVID-19 patients were compared with 184 historic controls. Inactive vitamin K-dependent MGP (desphospho-uncarboxylated [dp-uc] MGP) and prothrombin (PIVKA-II) were measured inversely related to extrahepatic and hepatic vitamin K status, respectively. Desmosine was measured to quantify the rate of elastic fiber degradation. Arterial calcification severity was assessed using computed tomography. RESULTS: dp-ucMGP was elevated in COVID-19 patients compared with controls (P < .001), with even higher dp-ucMGP in patients with poor outcomes (P < .001). PIVKA-II was normal in 82.1% of patients. dp-ucMGP was correlated with desmosine (P < .001) and with coronary artery (P = .002) and thoracic aortic (P < .001) calcification scores. CONCLUSIONS: dp-ucMGP was severely increased in COVID-19 patients, indicating extrahepatic vitamin K insufficiency, which was related to poor outcome; hepatic procoagulant factor II remained unaffected. These data suggest pneumonia-induced extrahepatic vitamin K depletion leading to accelerated elastic fiber damage and thrombosis in severe COVID-19 due to impaired activation of MGP and endothelial protein S, respectively.

Post hoc analysis of a randomised controlled trial: effect of vitamin D supplementation on circulating levels of desmosine in COPD.

BACKGROUND: Vitamin D supplementation lowers exacerbation frequency in severe vitamin D-deficient patients with COPD. Data regarding the effect of vitamin D on elastin degradation are lacking. Based on the vitamin's anti-inflammatory properties, we hypothesised that vitamin D supplementation reduces elastin degradation, particularly in vitamin D-deficient COPD patients. We assessed the effect of vitamin D status and supplementation on elastin degradation by measuring plasma desmosine, a biomarker of elastin degradation. METHODS: Desmosine was measured every 4 months in plasma of 142 vitamin D-naïve COPD patients from the Leuven vitamin D intervention trial (100 000 IU vitamin D3 supplementation every 4 weeks for 1 year). RESULTS: No significant association was found between baseline 25-hydroxyvitamin D (25(OH)D) and desmosine levels. No significant difference in desmosine change over time was found between the placebo and intervention group during the course of the trial. In the intervention arm, an unexpected inverse association was found between desmosine change and baseline 25(OH)D levels (p=0.005). CONCLUSIONS: Vitamin D supplementation did not have a significant overall effect on elastin degradation compared to placebo. Contrary to our hypothesis, the intervention decelerated elastin degradation in vitamin D-sufficient COPD patients and not in vitamin D-deficient subjects.

Time-course analysis of 3-epi-25-hydroxyvitamin D3 shows markedly elevated levels in early life, particularly from vitamin D supplementation in preterm infants.

BACKGROUND: An epimeric form of 25-hydroxyvitamin D3 (25(OH)D3) has recently been detected in clinical samples, with relatively high levels in infants. Little is known on 3-epi-25(OH)D3 formation and physiological function. Our objective was to study dynamics of 3-epi-25(OH)D3 formation during infancy. METHODS: 25(OH)D3 and 3-epi-25(OH)D3 levels were measured by liquid chromatography-tandem mass spectrometry in 22 preterm (aged 34-37 wk), 15 early preterm (aged <34 wk), and 118 term infants up to 2 y of age. All infants were prescribed vitamin D 400 IU/day after the first week of life. RESULTS: At birth, 3-epi-25(OH)D3 levels were 3 (1-7) nmol/l, <10% of total 25(OH)D3. From the second week to 3 mo of age, both 25(OH)D3 and 3-epi-25(OH)D3 increased, with highest 3-epi-25(OH)D3 contribution in early preterm infants (up to 55% of total 25(OH)D3 vs. 36% in term infants, P < 0.0001). After 3 mo of age, 3-epi-25(OH)D3 normalized to <10% in all infants. CONCLUSIONS: At birth, all infants showed low contribution of 3-epi-25(OH)D3, increasing the week after starting vitamin D supplementation, until 3 mo of age. Highest levels of 3-epi-25(OH)D3 were found in early preterm infants, supporting the hypothesis that hepatic immaturity plays a role in 3-epi-25(OH)D3 formation.

Overestimation of 25-hydroxyvitamin D3 by increased ionisation efficiency of 3-epi-25-hydroxyvitamin D3 in LC-MS/MS methods not separating both metabolites as determined by an LC-MS/MS method for separate quantification of 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum.

BACKGROUND: An LC-MS/MS method was developed for simultaneous quantification of 25-hydroxyvitamin D3 (25(OH)D3), 3-epi-25(OH)D3, and 25(OH)D2 in human serum. METHODS: Sample preparation consisted of protein precipitation followed by off-line SPE. Calibration curves for each vitamin D metabolite were constructed in phosphate-buffered saline with 60 g/L albumin including its corresponding stable isotope labelled (SIL) internal standard. A pentafluorophenyl (PFP) analytical column was used to resolve 25(OH)D3 from 25(OH)D2 and 3-epi-25(OH)D3, followed by SRM registration using positive ESI-MS/MS. Accuracy was assessed from measurement of samples with NIST reference method procedure (RMP) assigned values. The PFP LC-MS/MS method was compared to an in-house C18 column LC-MS/MS method, not resolving 25(OH)D3 from 3-epi-25(OH)D3, using adult and newborn samples. RESULTS: Intra-assay and inter-assay coefficients of variation were less than 4% and 7.5%, respectively for all three vitamin D metabolites; lower limits of quantification were 1, 1 and 2 nmol/L and linearity of methods were 1-500, 1-200 and 2-500 nmol/L for 25(OH)D3, 3-epi-25(OH)D3 and 25(OH)D2, respectively. The PFP LC-MS/MS method showed minimal bias to the NIST RMP. Method comparison revealed that in the C18 LC-MS/MS method, the 3-epi-25(OH)D3 concentration is overestimated inadvertently not only from co-elution of both analytes, but also by an additional 30-40% higher ionisation efficiency of 3-epi-25(OH)D3 when compared to 25(OH)D3. CONCLUSION: This accurate LC-MS/MS method allows the simultaneous measurement of 25(OH)D3, 3-epi-25(OH)D3, and 25(OH)D2 in human serum. Due to increased ionisation efficiency, the contribution of the 3-epi-25(OH)D3 metabolite to the total 25(OH)D3 concentration is significantly overestimated in MS methods that do not resolve 3-epi-25(OH)D3 from 25(OH)D3 and may compromise its use in infant samples known to have significant amounts of 3-epi-25(OH)D3.

Evaluation of 3-epi-25-hydroxyvitamin D3 cross-reactivity in the Roche Elecsys Vitamin D Total protein binding assay.

BACKGROUND: Presence of the 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3] metabolite affects accurate determination of 25(OH)D3 by most routine liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods and to an unknown extent in present immuno- and protein binding assays. We studied 3-epi-25(OH)D3 cross-reactivity in a competitive protein binding (CPB) assay (Roche Elecsys). METHODS: Neonatal samples, containing up to 58% of 3-epi-25(OH)D3 were used for measurement by the CPB assay and by an LC-MS/MS method separating 25(OH)D3 and 3-epi-25(OH)D3. Analytical recovery was also studied by addition of exogenous 3-epi-25(OH)D3. RESULTS: The CPB assay showed approximately 51% cross-reactivity to 3-epi-25(OH)D3 at exogenous addition. In contrast, there was minimal 3-epi-25(OH)D3 recognition by the CPB assay when present as the natural endogenous metabolite. CONCLUSIONS: The automated CPB assay displays minimal 3-epi-25(OH)D3 cross-reactivity in samples containing significant concentrations of endogenous 3-epi-25(OH)D3. Exogenous 3-epi-25(OH)D3 added to human serum or plasma seems to behave different from endogenous presence, and caution is warranted when using samples spiked with vitamin D metabolites for testing analytical specificity or external quality assurance in immuno- or protein binding assays.

Measurement of 25-OH-vitamin D in human serum using liquid chromatography tandem-mass spectrometry with comparison to radioimmunoassay and automated immunoassay.

The plasma 25-OH vitamin D concentration is a reliable biomarker for vitamin D status but assay's variability makes adequate monitoring of vitamin D status difficult. We employed isotope-dilution liquid chromatography (LC) tandem-mass spectrometry (MS/MS) for the measurement of both 25-OH vitamin D3 and 25-OH vitamin D2 in human serum. Hexadeuterium labelled 25-OH vitamin D3 internal standard (IS) was added to calibrators (prepared in phosphate-buffered saline with 60 g/L albumin), controls or patient sera and 25-OH vitamin D metabolites were released from vitamin D binding protein by adding sodium hydroxide prior to protein precipitation by acetonitrile/methanol (9:1, v/v). Subsequent off-line solid-phase extraction was followed by chromatographic separation on a C-18 column using a water/methanol/ammonium acetate gradient. Detection was by Atmospheric Pressure Electrospray Ionisation (AP-EI) followed by selected reaction monitoring. We compared the LC-MS/MS assay to the DiaSorin radioimmunoassay (RIA) and a recently re-standardised version of an automated electrochemiluminescent immunoassay (ECLIA) from Roche Diagnostics. We also analysed external quality control samples from the International Vitamin D External Quality Assessment Scheme (DEQAS) for comparison with other participating laboratories using LC-MS. The method was linear from 5 to at least 550 nmol/L with intra- and interday CV's < or = 6% for both 25-OH vitamin D3 and 25-OH vitamin D2. Recoveries ranged between 94.9 and 106.9% for 25-OH vitamin D3 and 82.7 and 100.3% for 25-OH vitamin D2. Our results for the DEQAS serum pools averaged -7.2% from the overall LC-MS method mean. The DiaSorin RIA agreed well with the LC-MS/MS method (r(2)=0.90; average bias 1.61 nmol/L), the Roche ECLIA considerably disagreed (r(2)=0.58; bias 10.13 nmol/L). This LC-MS/MS method is reliable and robust for the measurement of both 25-OH vitamin D3 and 25-OH vitamin D2 in human serum.

Unanticipated error in HbA(1c) measurement on the HLC-723 G7 analyzer.

OBJECTIVES: Investigation of falsely elevated HbA(1c) measurements on the HLC-723 G7 analyser. DESIGN AND METHODS: Comparison of HbA(1c) in blood samples that were diluted either in hemolysis reagent or water. RESULTS: HbA(1c) results became falsely elevated when samples were diluted in hemolysis reagent, but not in water. QC-procedures failed to detect this error as calibrator and QC samples were manually diluted in water, according to manufacturer's instructions, whereas patient samples were automatically diluted using hemolysing reagent. After replacement of the instruments' sample-loop and rotor seal comparable HbA(1c) results were obtained, irrespective of dilution with hemolysing reagent or water. CONCLUSION: This case illustrates the importance of treating calibrator and QC materials similar to routine patient samples in order to prevent unnoticed drift in patient HbA(1c) results.

Plasma citrulline measurement using UPLC tandem mass-spectrometry to determine small intestinal enterocyte pathology.

Citrulline is a nonessential free amino acid, detectable in various biological fluids such as plasma, urine and cerebrospinal fluid. The plasma citrulline concentration is increasingly considered to be a reliable biomarker of enterocyte function. Current analysis usually involves lengthy HPLC separations as a part of classical amino acid profiling, or mass spectrometry usually in combination with derivatization. We employed UPLC-HILIC-tandem mass-spectrometry (MS/MS) of acetonitrile-derived supernatants from plasma samples of control subjects and of patients who had received myeloablative chemotherapy. Detection was achieved by the selected reaction monitoring of transitions: m/z 176-->70 and 180-->74 (for the deuterated standard), respectively. The method was precise and accurate with inter-day CV<3.9% (n=30), recoveries ranging from 98.0 to 100.3% and high linearity from 0.3 to at least 2,000 micromol/L. The results for 202 plasma samples agreed well with those obtained by the classical HPLC-fluorescence method. By a simple protein precipitation/extraction step and the UPLC separation the result can be available within 30 min of receipt with a capacity of at least 12 assays per hour. Citrulline in blood and plasma or serum was stable for at least 2 days at room temperature which would permit postal transport to the laboratory. The UPLC-MS/MS method for measuring plasma citrulline concentrations is fast and robust and is therefore an ideal tool for monitoring the intestinal enterocyte capacity of patients with various pathological conditions.

The silent hemoglobin alpha chain variant Hb Riccarton [alpha51(CE9)Gly-->Ser] may affect HbA1c determination on the HLC-723 G7 analyzer.

BACKGROUND: Structural hemoglobin variants can affect the accuracy of hemoglobin A1c (HbA1c) testing and represent the most common pitfall in the determination of HbA1c. We here describe the characterization of an alpha chain variant in diabetic patients as the cause of an abnormal presentation of the HbA1c fraction on the HLC-723 G7 analyzer. METHODS: HbA1c analysis was performed using various HPLC-based HbA1c analyzers and by immunoassay. alpha-Globin mutation analysis was performed by GAP-PCR and DNA sequencing. RESULTS: The peak partially overlapping HbA1c in the chromatogram represents the glycated fraction of the silent alpha chain variant Hb Riccarton [alpha51(CE9)Gly-->Ser]. This aberrant peak is uniquely identified by the HLC-723 instrument, as it is not observed on other HPLC-based HbA1c analyzers. Occasionally, the HLC-723 may fail to properly integrate both glycated Hb fractions, resulting in a falsely low HbA1c result. The variant was confirmed in samples from other diabetic patients with identical chromatographic patterns. CONCLUSIONS: The silent alpha chain variant Hb Riccarton [alpha51(CE9)Gly-->Ser] leads to an abnormal chromatographic presentation on the HLC-723 analyzer with a risk of erroneous HbA1c determination. Manual validation of chromatograms to detect abnormalities caused by Hb variants is important to prevent incorrectly produced HbA1c results from being reported.

Validation study of Axis-Shield carbohydrate-deficient transferrin (CDT) on ARCHITECT chemistry analyzer.

BACKGROUND: Carbohydrate-deficient transferrin (CDT) is a biochemical marker used for identifying chronic alcohol intake. We developed and validated an ARCHITECT c8000 (Abbott) instrument application for the Axis-Shield %CDT immunoassay. METHODS: Standard CLSI (Clinical and Laboratory Standards Institute) evaluation protocols were performed. RESULTS: The Axis-Shield %CDT ARCHITECT method was standardized against the Axis-Shield %CDT microtiter test by linear regression analysis (n=50 mean of duplicate, R=0.996). Method comparison by Deming regression revealed a slope of 1.01 and an intercept of -0.03 with Axis-Shield %CDT microtiter test (n=50 in duplicate, R=0.995), and a slope of 0.82 and an intercept of 1.09 with HPLC method (n=47 in duplicate, R=0.990) as the candidate IFCC (International Federation of Clinical Chemistry and Laboratory Medicine) reference method. The predicted medical decision points (MDPs) are both 2.6% and equal the MDP that is generally used for the Axis-Shield %CDT tests. Precision derived from pooled patient sera (low level) and commercially available control material (high level) was excellent. Total variation was 3.2% and 1.8%, respectively. CONCLUSIONS: The Axis-Shield %CDT ARCHITECT method, as one of the first Axis-Shield applications on a large-scale analyzer, is a reliable test for routine %CDT analysis providing precise and well-standardized %CDT results.