Ido P. Kema

Accuracy of 6 Routine 25-Hydroxyvitamin D Assays: Influence of Vitamin D Binding Protein Concentration

BACKGROUNDnRecent recognition of its broad pathophysiological importance has triggered an increased interest in 25-hydroxyvitamin D [25(OH)D]. By consequence, throughput in 25(OH)D testing has become an issue for clinical laboratories, and several automated assays for measurement of 25(OH)D are now available. The aim of this study was to test the accuracy and robustness of these assays by comparing their results to those of an isotope dilution/online solid-phase extraction liquid chromatography/tandem mass spectrometry (ID-XLC-MS/MS) method. We put specific focus on the influence of vitamin D-binding protein (DBP) by using samples with various concentrations of DBP.nnnMETHODSnWe used 5 automated assays (Architect, Centaur, iSYS, Liaison, and Elecsys), 1 RIA (Diasorin) preceded by extraction, and an ID-XLC-MS/MS method to measure 25(OH)D concentrations in plasma samples of 51 healthy individuals, 52 pregnant women, 50 hemodialysis patients, and 50 intensive care patients. Using ELISA, we also measured DBP concentrations in these samples.nnnRESULTSnMost of the examined 25(OH)D assays showed significant deviations in 25(OH)D concentrations from those of the ID-XLC-MS/MS method. As expected, DBP concentrations were higher in samples of pregnant women and lower in samples of IC patients compared to healthy controls. In 4 of the 5 fully automated 25(OH)D assays, we observed an inverse relationship between DBP concentrations and deviations from the ID-XLC-MS/MS results.nnnCONCLUSIONSn25(OH)D measurements performed with most immunoassays suffer from inaccuracies that are DBP concentration dependent. Therefore, when interpreting results of 25(OH)D measurements, careful consideration of the measurement method is necessary.

Traditionally living populations in East Africa have a mean serum 25-hydroxyvitamin D concentration of 115 nmol/l.

Cutaneous synthesis of vitamin D by exposure to UVB is the principal source of vitamin D in the human body. Our current clothing habits and reduced time spent outdoors put us at risk of many insufficiency-related diseases that are associated with calcaemic and non-calcaemic functions of vitamin D. Populations with traditional lifestyles having lifelong, year-round exposure to tropical sunlight might provide us with information on optimal vitamin D status from an evolutionary perspective. We measured the sum of serum 25-hydroxyvitamin D₂ and D₃ (25(OH)D) concentrations of thirty-five pastoral Maasai (34 (SD 10) years, 43 % male) and twenty-five Hadzabe hunter-gatherers (35 (SD 12) years, 84 % male) living in Tanzania. They have skin type VI, have a moderate degree of clothing, spend the major part of the day outdoors, but avoid direct exposure to sunlight when possible. Their 25(OH)D concentrations were measured by liquid chromatography-MS/MS. The mean serum 25(OH)D concentrations of Maasai and Hadzabe were 119 (range 58-167) and 109 (range 71-171) nmol/l, respectively. These concentrations were not related to age, sex or BMI. People with traditional lifestyles, living in the cradle of mankind, have a mean circulating 25(OH)D concentration of 115 nmol/l. Whether this concentration is optimal under the conditions of the current Western lifestyle is uncertain, and should as a possible target be investigated with concomitant appreciation of other important factors in Ca homeostasis that we have changed since the agricultural revolution.

The role of liquid chromatography-tandem mass spectrometry in the clinical laboratory.

Liquid chromatography coupled to mass spectrometry (LC-MS/MS) is increasingly used as a routine methodology in clinical laboratories for the analysis of low molecular weight molecules. The high specificity in combination with high sensitivity and multi-analyte potential makes it an attractive complementary method to traditional methodology used for routine applications. Its strength and weaknesses in this context will be discussed and examples of successful clinical applications will be given. For LC-MS/MS to truly fulfil its promise in clinical diagnosis, the prerequisite steps being sample pre-treatment, chromatographic separation and detection by selected reaction monitoring must become more integrated as they are in conventional clinical analysers. The availability of ready-to-use reagents kits, eliminating efforts needed for method development and extensive validation, are likely to contribute to a wider acceptance of LC-MS/MS in clinical laboratories. Growing applicability of LC-MS/MS in the clinical laboratory field is expected from quantitative protein analysis.

Delaying aging and the aging-associated decline in protein homeostasis by inhibition of tryptophan degradation

Toxicity of aggregation-prone proteins is thought to play an important role in aging and age-related neurological diseases like Parkinson and Alzheimer’s diseases. Here, we identify tryptophan 2,3-dioxygenase (tdo-2), the first enzyme in the kynurenine pathway of tryptophan degradation, as a metabolic regulator of age-related α-synuclein toxicity in a Caenorhabditis elegans model. Depletion of tdo-2 also suppresses toxicity of other heterologous aggregation-prone proteins, including amyloid-β and polyglutamine proteins, and endogenous metastable proteins that are sensors of normal protein homeostasis. This finding suggests that tdo-2 functions as a general regulator of protein homeostasis. Analysis of metabolite levels in C. elegans strains with mutations in enzymes that act downstream of tdo-2 indicates that this suppression of toxicity is independent of downstream metabolites in the kynurenine pathway. Depletion of tdo-2 increases tryptophan levels, and feeding worms with extra l-tryptophan also suppresses toxicity, suggesting that tdo-2 regulates proteotoxicity through tryptophan. Depletion of tdo-2 extends lifespan in these worms. Together, these results implicate tdo-2 as a metabolic switch of age-related protein homeostasis and lifespan. With TDO and Indoleamine 2,3-dioxygenase as evolutionarily conserved human orthologs of TDO-2, intervening with tryptophan metabolism may offer avenues to reducing proteotoxicity in aging and age-related diseases.

Quantitative profiling of tryptophan metabolites in serum, urine, and cell culture supernatants by liquid chromatography-tandem mass spectrometry

A sensitive, selective, and comprehensive method for the quantitative determination of tryptophan and 18 of its key metabolites in serum, urine, and cell culture supernatants was developed. The analytes were separated on a C18 silica column by reversed-phase liquid chromatography and detected by electrospray ionization tandem mass spectrometry in positive ion multiple reaction monitoring (MRM) mode, except for indoxyl sulfate which was measured in negative ion MRM mode in a separate run. The limits of detection and lower limits of quantification were in the range of 0.1–50 and 0.5–100xa0nM, respectively. Fully 13C isotope-labeled and deuterated internal standards were used to achieve accurate quantification. The applicability of the method to analyze serum, urine, and cell culture supernatants was demonstrated by recovery experiments and the evaluation of matrix effects. Precision for the analysis of serum, urine, and cell culture supernatants ranged between 1.3% and 16.0%, 1.5% and 13.5%, and 1.0% and 17.4%, respectively. The method was applied to analyze changes in tryptophan metabolism in cell culture supernatants from IFN-γ-treated monocytes and immature or mature dendritic cells.

Vitamin D status indicators in indigenous populations in East Africa

PurposeSufficient vitamin D status may be defined as the evolutionary established circulating 25-hydroxyvitamin D [25(OH)D] matching our Paleolithic genome.MethodsWe studied serum 25(OH)D [defined as 25(OH)D2xa0+xa025(OH)D3] and its determinants in 5 East African ethnical groups across the life cycle: Maasai (MA) and Hadzabe (HA) with traditional life styles and low fish intakes, and people from Same (SA; intermediate fish), Sengerema (SE; high fish), and Ukerewe (UK; high fish). Samples derived from non-pregnant adults (MA, HA, SE), pregnant women (MA, SA, SE), mother–infant couples at delivery (UK), infants at delivery and their lactating mothers at 3xa0days (MA, SA, SE), and lactating mothers at 3xa0months postpartum (SA, SE). Erythrocyte docosahexaenoic acid (RBC-DHA) was determined as a proxy for fish intake.ResultsThe meanxa0±xa0SD 25(OH)D of non-pregnant adults and cord serum were 106.8xa0±xa028.4 and 79.9xa0±xa026.4xa0nmol/L, respectively. Pregnancy, delivery, ethnicity (which we used as a proxy for sunlight exposure), RBC-DHA, and age were the determinants of 25(OH)D. 25(OH)D increased slightly with age. RBC-DHA was positively related to 25(OH)D, notably 25(OH)D2. Pregnant MA (147.7 vs. 118.3) and SE (141.9 vs. 89.0) had higher 25(OH)D than non-pregnant counterparts (MA, SE). Infant 25(OH)D at delivery in Ukerewe was about 65xa0% of maternal 25(OH)D.ConclusionsOur ancient 25(OH)D amounted to about 115xa0nmol/L and sunlight exposure, rather than fish intake, was the principal determinant. The fetoplacental unit was exposed to high 25(OH)D, possibly by maternal vitamin D mobilization from adipose tissue, reduced insulin sensitivity, trapping by vitamin D-binding protein, diminished deactivation, or some combination.

Short-term vitamin D3 supplementation lowers plasma renin activity in patients with stable chronic heart failure: An open-label, blinded end point, randomized prospective trial (VitD-CHF trial)

BACKGROUNDnMany chronic heart failure (CHF) patients have low vitamin D (VitD) and high plasma renin activity (PRA), which are both associated with poor prognosis. Vitamin D may inhibit renin transcription and lower PRA. We investigated whether vitamin D3 (VitD3) supplementation lowers PRA in CHF patients.nnnMETHODS AND RESULTSnWe conducted a single-center, open-label, blinded end point trial in 101 stable CHF patients with reduced left ventricular ejection fraction. Patients were randomized to 6 weeks of 2,000 IU oral VitD3 daily or control. At baseline, mean age was 64 ± 10 years, 93% male, left ventricular ejection fraction 35% ± 8%, and 56% had VitD deficiency. The geometric mean (95% CI) of 25-hydroxyvitamin D3 increased from 48 nmol/L (43-54) at baseline to 80 nmol/L (75-87) after 6 weeks in the VitD3 treatment group and decreased from 47 nmol/L (42-53) to 44 nmol/L (39-49) in the control group (P < .001). The primary outcome PRA decreased from 6.5 ng/mL per hour (3.8-11.2) to 5.2 ng/mL per hour (2.9-9.5) in the VitD3 treatment group and increased from 4.9 ng/mL per hour (2.9-8.5) to 7.3 ng/mL per hour (4.5-11.8) in the control group (P = .002). This was paralleled by a larger decrease in plasma renin concentration in the VitD3 treatment group compared to control (P = .020). No significant changes were observed in secondary outcome parameters, including N-terminal pro-B-type natriuretic peptide natriuretic peptide and fibrosis markers.nnnCONCLUSIONSnMost CHF patients had VitD deficiency and high PRA levels. Six weeks of supplementation with 2,000 IU VitD3 increased 25-hydroxyvitamin D3 levels and decreased PRA and plasma renin concentration.

Discovery and fine mapping of serum protein loci through transethnic meta-analysis

Many disorders are associated with altered serum protein concentrations, including malnutrition, cancer, and cardiovascular, kidney, and inflammatory diseases. Although these protein concentrations are highly heritable, relatively little is known about their underlying genetic determinants. Through transethnic meta-analysis of European-ancestry and Japanese genome-wide association studies, we identified six loci at genome-wide significance (p < 5xa0× 10(-8)) for serum albumin (HPN-SCN1B, GCKR-FNDC4, SERPINF2-WDR81, TNFRSF11A-ZCCHC2, FRMD5-WDR76, and RPS11-FCGRT, in up to 53,190 European-ancestry and 9,380 Japanese individuals) and three loci for total protein (TNFRS13B, 6q21.3, and ELL2, in up to 25,539 European-ancestry and 10,168 Japanese individuals). We observed little evidence of heterogeneity in allelic effects at these loci between groups of European and Japanese ancestry but obtained substantial improvements in the resolution of fine mapping of potential causal variants by leveraging transethnic differences in the distribution of linkage disequilibrium. We demonstrated a functional role for the most strongly associated serum albumin locus, HPN, for which Hpn knockout mice manifest low plasma albumin concentrations. Other loci associated with serum albumin harbor genes related to ribosome function, protein translation, and proteasomal degradation, whereas those associated with serum total protein include genes related to immune function. Our results highlight the advantages of transethnic meta-analysis for the discovery and fine mapping of complex trait loci and have provided initial insights into the underlying genetic architecture of serum protein concentrations and their association with human disease.

Current status and future developments of LC-MS/MS in clinical chemistry for quantification of biogenic amines

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is rapidly gaining ground in special clinical chemistry laboratories. It significantly increases the analytic potential in clinical chemistry, especially in the field of low molecular weight biomarker analysis. This review overviews current LC-MS/MS methods used for the quantification of biogenic amines and their metabolites. New possibilities offered by this technique are illustrated by recently developed assays for biogenic amines. Major shortcomings of conventional chromatographic techniques, such as labor-intensive sample preparation, long analysis times and often the relatively low specificity, are circumvented by using LC-MS/MS. In addition, LC-MS/MS has broad analyte compatibility and high analytical performance. In the last 5 years introduction of LC-MS/MS in routine diagnostics has resulted in improved assays for diagnosis and follow-up of neuroendocrine tumors characterized by the secretion of biogenic amines. Due to their labile nature and low concentration ranges biogenic amines require extensive and careful sample preparation. Introduction of new sophisticated techniques such as selective sorbents adsorption is evolving. This enables not only more specific analyte selection, but also automation of the complicated clean-up procedure. Automated sample clean-up can be directly coupled to LC-MS/MS, which facilitates reproducible and efficient handling of the growing number of samples to be analyzed in laboratories.

Distinct Tryptophan Catabolism and Th17/Treg Balance in HIV Progressors and Elite Controllers

Tryptophan (Trp) catabolism into immunosuppressive kynurenine (Kyn) by indoleamine 2,3-dioxygenase (IDO) was previously linked to Th17/Treg differentiation and immune activation. Here we examined Trp catabolism and its impact on Th17/Treg balance in uninfected healthy subjects (HS) and a large cohort of HIV-infected patients with different clinical outcomes: ART-naïve, Successfully Treated (ST), and elite controllers (EC). In ART-naïve patients, increased IDO activity/expression, together with elevated levels of TNF-α and sCD40L, were associated with Treg expansion and an altered Th17/Treg balance. These alterations were normalized under ART. In contrast, Trp 2,3-dioxegenase (TDO) expression was dramatically lower in EC when compared to all other groups. Interestingly, EC displayed a distinctive Trp metabolism characterized by low Trp plasma levels similar to ART-naïve patients without accumulating immunosuppressive Kyn levels which was accompanied by a preserved Th17/Treg balance. These results suggest a distinctive Trp catabolism and Th17/Treg balance in HIV progressors and EC. Thus, IDO-induced immune-metabolism may be considered as a new inflammation-related marker for HIV-1 disease progression.