Exploring the kynurenine pathway in mild traumatic brain injury: A longitudinal study.

A potential source of novel biomarkers for mTBI is the kynurenine pathway (KP), a metabolic pathway of tryptophan (Trp), that is up-regulated by neuroinflammation and stress. Considering that metabolites of the KP (kynurenines) are implicated in various neuropsychiatric diseases, exploration of this pathway could potentially bridge the gap between physiological and psychological factors in the recovery process after mTBI. This study, therefore, set out to characterize the KP after mTBI and to examine associations with long-term outcome. Patients were prospectively recruited at the emergency department (ED), and blood samples were obtained in the acute phase (<24 h; N = 256) and at 1-month follow-up (N = 146). A comparison group of healthy controls (HC; N = 32) was studied at both timepoints. Trp, kynurenines, and interleukin (IL)-6 and IL-10 were quantified in plasma. Clinical outcome was measured at six months post-injury. Trp, xanthurenic acid (XA), and picolinic acid (PA) were significantly reduced in patients with mTBI relative to HC, corrected for age and sex. For Trp (d = -0.57 vs. d = -0.29) and XA (d = -0.98 vs. d = -0.32), larger effects sizes were observed during the acute phase compared to one-month follow-up, while for PA (d = -0.49 vs. d = -0.52) effect sizes remained consistent. Findings for other kynurenines (e.g., kynurenine, kynurenic acid, and quinolinic acid) were non-significant after correction for multiple testing. Within the mTBI group, lower acute Trp levels were significantly related to incomplete functional recovery and higher depression scores at 6 months post-injury. No significant relationships were found for Trp, XA, and PA with IL-6 or IL-10 concentrations. In conclusion, our findings indicate that perturbations of the plasma KP in the hyperacute phase of mTBI and 1 month later are limited to the precursor Trp, and glutamate system modulating kynurenines XA and PA. Correlations between acute reductions of Trp and unfavorable outcomes may suggest a potential substrate for pharmacological intervention.

Hereditary Alpha Tryptasemia: Validation of a Single-Well Multiplex Digital Droplet PCR Assay in a Cohort of Symptomatic Patients.

BACKGROUND: Hereditary alpha tryptasemia (HαT) has significant prevalence and potential morbidity in the general population. However, it remains largely undiagnosed in routine clinical diagnostics due to low availability of efficient assessment methods. To address this issue, we developed a reliable and efficient single-well multiplex digital droplet PCR assay. METHODS: The assay was based on the reconstruction of the TPSAB1 gene through quantification of the ratio of α- and ß-tryptase copy number variants (CNV) in a single-well measurement. We performed analytical validation by determining CNV measurement clustering around the expected copy numbers in 281 cases and determined the diagnostic accuracy of basal serum tryptase (BST) to predict HαT and HαT subtypes in 141 symptomatic patients. RESULTS: The assay determined α- and ß-tryptase CNVs with an overall accuracy, expressed as a 99% prediction interval, of 0.03 ± 0.27 copy numbers. The optimal BST cutoff level to predict HαT in symptomatic patients, who had no other explanation for relatively high tryptase levels (i.e., no diagnosis of systemic mastocytosis, myeloid neoplasm, or end-stage renal failure), was 9.2 ng/mL (sensitivity: 98.1%; specificity: 96.6%). HαT showed a linear gene-dose effect, with an average gene-dose increase of 7.5 ng/mL per extra α-tryptase gene. CONCLUSION: Our single-well multiplex digital droplet PCR assay accurately determined HαT and could be implemented as a state-of-the-art routine diagnostic test. The assay demonstrated a strong correlation with BST and the optimal threshold for identifying HαT in symptomatic patients with unexplained high tryptase concentrations was at a BST level of 9.2 ng/mL.

Creatine homeostasis and the kidney: comparison between kidney transplant recipients and healthy controls.

Creatine is a natural nitrogenous organic acid that is integral to energy metabolism and crucial for proper cell functioning. The kidneys are involved in the first step of creatine production. With kidney transplantation being the gold-standard treatment for end-stage kidney disease, kidney transplant recipients (KTR) may be at risk of impaired creatine synthesis. We aimed to compare creatine homeostasis between KTR and controls. Plasma and urine concentrations of arginine, glycine, guanidinoacetate, creatine and creatinine were measured in 553 KTR and 168 healthy controls. Creatine intake was assessed using food frequency questionnaires. Iothalamate-measured GFR data were available in subsets of 157 KTR and 167 controls. KTR and controls had comparable body weight, height and creatine intake (all P > 0.05). However, the total creatine pool was 14% lower in KTR as compared to controls (651 ± 178 vs. 753 ± 239 mmol, P < 0.001). The endogenous creatine synthesis rate was 22% lower in KTR as compared to controls (7.8 ± 3.0 vs. 10.0 ± 4.1 mmol per day, P < 0.001). Despite lower GFR, the plasma guanidinoacetate and creatine concentrations were 21% and 41% lower in KTR as compared to controls (both P < 0.001). Urinary excretion of guanidinoacetate and creatine were 66% and 59% lower in KTR as compared to controls (both P < 0.001). In KTR, but not in controls, a higher measured GFR was associated with a higher endogenous creatine synthesis rate (std. beta: 0.21, 95% CI: 0.08; 0.33; P = 0.002), as well as a higher total creatine pool (std. beta: 0.22, 95% CI: 0.11; 0.33; P < 0.001). These associations were fully mediated (93% and 95%; P < 0.001) by urinary guanidinoacetate excretion which is consistent with production of the creatine precursor guanidinoacetate as rate-limiting factor. Our findings highlight that KTR have a disturbed creatine homeostasis as compared to controls. Given the direct relationship of measured GFR with endogenous creatine synthesis rate and the total creatine pool, creatine supplementation might be beneficial in KTR with low kidney function.Trial registration ID: NCT02811835.Trial registration URL: https://clinicaltrials.gov/ct2/show/NCT02811835 .

Discriminatory Value of Steroid Hormones on Polycystic Ovary Syndrome and Clustering of Hyperandrogenism and Metabolic Factors.

OBJECTIVE: We determined (1) if 11-oxygenated androgens better identify polycystic ovary syndrome (PCOS) diagnosis in women with obesity compared to total or free testosterone (T) and free androgen index; (2) how biochemical hyperandrogenism and metabolic factors cluster in a cohort of women with infertility and obesity. METHODS: Women with obesity and PCOS comprised the study group (N = 132). Ovulatory women with obesity and idiopathic, tubal or male factor infertility were the control group (N = 83). Steroid hormones were measured by means of liquid chromatography tandem mass spectrometry. Receiver operating characteristic curves and principal component analysis were used. RESULTS: Women with obesity and PCOS had higher 11-ketotestosterone (11 KT) (1.22 nmol/L [0.84; 1.65] vs 1.05 [0.78; 1.35], P = .04) compared to controls, but not 11ß-hydroxyandrostenedione 4.30 [2.87; 5.92] vs 4.06 [3.22; 5.73], P = .44). 11-ketotestosterone (area under the curve: 0.59) did not better discriminate PCOS in women with obesity compared to: total T (0.84), free T (0.91), and free androgen index (0.85). We identified 4 principal components (PCs) in the PCOS group (72.1% explained variance): (1) insulin resistance status; (2) blood pressure; (3) obesity; (4) androgen status and 4 PCs in the control group (68.7% explained variance) with variables representing metabolism being dispersed in component 2, 3, and 4. CONCLUSIONS: Eleven-oxygenated androgens do not aid in the diagnosis of PCOS in women with obesity. Insulin resistance is the strongest PC in the PCOS group. There is no major dominant characteristic that defines obese non-PCOS women.

Sleep quality, fatigue, societal participation and health-related quality of life in kidney transplant recipients: a cross-sectional and longitudinal cohort study.

BACKGROUND: Fatigue and impaired health-related quality of life (HRQoL) are common among kidney transplant recipients (KTR). We hypothesized that both may partially be attributable to poor sleep. METHODS: Cross-sectional and longitudinal data of KTR enrolled in the TransplantLines Biobank and Cohort Study were used. Sleep quality was assessed using the Pittsburgh Sleep Quality Index questionnaire. Individual strength (i.e. a composite of fatigue, concentration, motivation and physical activity), societal participation and HRQoL were assessed using validated questionnaires. RESULTS: We included 872 KTR (39% female, age 56 ± 13 years) and 335 healthy controls. In total, 33% of male KTR and 49% of female KTR reported poor sleep quality, which was higher compared with male and female healthy controls (19% and 28%, respectively, P < .001 for both). In logistic regression analyses, female sex, anxiety, active smoking, low protein intake, physically inactive lifestyle, low plasma magnesium concentration, using calcineurin inhibitors, not using mTOR inhibitors and using benzodiazepine agonists were associated with poor sleep quality. In adjusted linear regression analyses, poor sleep was strongly and independently associated with lower individual strength [standardized ß (st.ß) = 0.59, 95% confidence interval (CI) 0.45 to 0.74, P < .001], poorer societal participation (frequency: st.ß = -0.17, 95% CI -0.32 to -0.01, P = .04; restrictions: st.ß = -0.36, 95% CI -0.51 to -0.21, P < .001; satisfaction: st.ß = -0.44, 95% CI -0.59 to -0.28, P < .001) and lower HRQoL (physical: st.ß = -0.53, 95% CI -0.68 to -0.38, P < .001; mental: st.ß = -0.64, 95% CI -0.78 to -0.50, P < .001). The associations with poorer societal participation and lower HRQoL were strongly mediated by individual strength (P < .001 for all), yet the suggested direct effects of poor sleep quality on HRQoL remained significant (Pphysical = .03, Pmental = .002). Longitudinal data of 292 KTR showed that sleep quality improves after kidney transplantation in males (P < .001), but not in females (P = .9). CONCLUSIONS: Poor sleep quality is common among KTR, and may be a potential target to improve fatigue, societal participation and HRQoL among KTR.

Dietary lithium intake, graft failure and mortality in kidney transplant recipients.

BACKGROUND: Long-term high-dose lithium therapy in bipolar disorder is known to adversely affect kidney function. However, recent animal studies have revealed that low amounts of lithium are beneficial for the kidney when it is damaged by exposure to nephrotoxic compounds, inflammation or oxidative stress. This study aimed to investigate whether urinary lithium excretion, reflecting dietary lithium intake, is associated with adverse long-term kidney graft outcomes and patient survival. METHODS: Urinary lithium concentration was measured using inductively coupled plasma mass spectrometry in 642 stable kidney transplant recipients (KTRs). Graft failure was defined as the start of dialysis or retransplantation and kidney function decline was defined as a doubling of serum creatinine. RESULTS: The median urinary lithium excretion was 3.03 µmol/24 h [interquartile range (IQR) 2.31-4.01]. Urinary lithium excretion was associated with energy, plant protein and water intake. During a median follow-up of 5.3 years (IQR 4.5-6.0), 79 (12%) KTRs developed graft failure and 127 (20%) KTRs developed kidney function decline. Higher urinary lithium excretion was associated with a lower risk of graft failure {hazard ratio [HR] per doubling 0.54 [95% confidence interval (CI) 0.38-0.79]} and kidney function decline [HR per doubling 0.73 (95% CI 0.54-0.99)]. These associations remained independent of adjustment for potential confounders and in sensitivity analyses. There was a significant effect modification with the use of proliferation inhibitors (P = .05) and baseline estimated glomerular filtration rate (eGFR; P < .001), with higher urinary lithium excretion being more protective in KTRs not using proliferation inhibitors and in KTRs with lower baseline eGFR. Furthermore, higher urinary lithium excretion was associated with a reduced risk of all-cause mortality [HR 0.64 (95% CI 0.49-0.83); P = .001]. CONCLUSION: Dietary lithium intake may be a potentially modifiable, yet rather overlooked, risk factor for adverse long-term kidney graft outcomes and patient survival. TRIAL REGISTRATION: https://clinicaltrials.gov/ct2/show/NCT02811835.

An antibody-free LC-MS/MS method for the quantification of sex hormone binding globulin in human serum and plasma.

OBJECTIVES: Sex hormone binding globulin (SHBG) is a hormone binding protein which plays an important role in regulating the transport and availability of biologically active androgens and estradiol to target cells and used to calculate free testosterone concentrations. METHODS: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed, featuring an albumin removal step followed by a tryptic digestion. After a reduction step with dithiothreitol and alkylation with iodoacetamide three signature peptides were used for the quantification of SHBG. RESULTS: The method enables the quantification of serum and plasma SHBG over the clinically relevant range of 200-20,000 ng/mL and was validated according to the most recent guidelines. The LC-MS/MS method correlates well with the Abbott Alinity immunoassay (R2>0.95), but the LC-MS/MS results are on average 16-17% lower than the immunoassay results, which is consistent for all three signature peptides. CONCLUSIONS: The LC-MS/MS method which includes an albumin depletion step allows quantification of SHBG in serum and plasma without an immunocapture step at clinically relevant SHBG levels, thus contributing to better lab-to-lab consistency of results.

Elevated cerebrospinal fluid glucose levels and diabetes mellitus are associated with activation of the neurotoxic polyol pathway.

AIMS/HYPOTHESIS: During hyperglycaemia, some glucose bypasses glycolysis and is metabolised via the potentially neurotoxic polyol pathway, in which glucose is metabolised to sorbitol and fructose. Increased polyol concentrations have been demonstrated in the cerebrospinal fluid (CSF) of neurological patients with and without diabetes mellitus. However, polyol levels in patients without evident neurological abnormalities have not been investigated so far. The aim of this study was to determine CSF polyol concentrations in patients without major neurological disease with normal or elevated CSF glucose concentrations. METHODS: This observational cohort study used CSF and plasma analyses, as well as clinical data, from 30 participants of the Anaesthetic Biobank of Cerebrospinal Fluid study. Biomaterial was collected from adult patients scheduled for elective surgery under spinal anaesthesia. CSF polyol concentrations were measured by GC/flame ionisation detector in ten patients with normal CSF glucose levels (group 1), ten patients with elevated CSF glucose levels (group 2) and ten patients with elevated CSF glucose levels and type 2 diabetes (group 3). We compared the concentrations of plasma glucose, CSF glucose, sorbitol and fructose, and CSF polyol/glucose ratios between the three groups, and determined the correlation between plasma glucose levels and CSF glucose, sorbitol and fructose levels. RESULTS: Groups 2 and 3 had significantly higher CSF fructose levels compared with group 1 (p=0.036 and p<0.001, respectively). Group 3 showed significant differences compared with groups 1 and 2 for CSF sorbitol (p<0.001 and 0.036, respectively). Moreover, patients with diabetes had a significantly higher CSF sorbitol/glucose ratio compared with patients without diabetes. There was a strong positive correlation between plasma glucose and CSF glucose, sorbitol and fructose. Finally, age, sex, CSF/plasma albumin ratio and preoperative cognitive function scores were significantly correlated with plasma glucose and CSF glucose, sorbitol and fructose levels. CONCLUSIONS/INTERPRETATION: Hyperglycaemia causes a proportional increase in polyol concentrations in CSF of patients without major neurological disease. Furthermore, this study provides the first indication of upregulation of the cerebral polyol pathway in patients with diabetes without evident neurological abnormalities.

A novel mouse model for pyridoxine-dependent epilepsy due to antiquitin deficiency.

Pyridoxine-dependent epilepsy (PDE) is a rare autosomal recessive disease caused by mutations in the ALDH7A1 gene leading to blockade of the lysine catabolism pathway. PDE is characterized by recurrent seizures that are resistant to conventional anticonvulsant treatment but are well-controlled by pyridoxine (PN). Most PDE patients also suffer from neurodevelopmental deficits despite adequate seizure control with PN. To investigate potential pathophysiological mechanisms associated with ALDH7A1 deficiency, we generated a transgenic mouse strain with constitutive genetic ablation of Aldh7a1. We undertook extensive biochemical characterization of Aldh7a1-KO mice consuming a low lysine/high PN diet. Results showed that KO mice accumulated high concentrations of upstream lysine metabolites including ∆1-piperideine-6-carboxylic acid (P6C), α-aminoadipic semialdehyde (α-AASA) and pipecolic acid both in brain and liver tissues, similar to the biochemical picture in ALDH7A1-deficient patients. We also observed preliminary evidence of a widely deranged amino acid profile and increased levels of methionine sulfoxide, an oxidative stress biomarker, in the brains of KO mice, suggesting that increased oxidative stress may be a novel pathobiochemical mechanism in ALDH7A1 deficiency. KO mice lacked epileptic seizures when fed a low lysine/high PN diet. Switching mice to a high lysine/low PN diet led to vigorous seizures and a quick death in KO mice. Treatment with PN controlled seizures and improved survival of high-lysine/low PN fed KO mice. This study expands the spectrum of biochemical abnormalities that may be associated with ALDH7A1 deficiency and provides a proof-of-concept for the utility of the model to study PDE pathophysiology and to test new therapeutics.

Selective quantification of the 22-kDa isoform of human growth hormone 1 in serum and plasma by immunocapture and LC-MS/MS.

The human growth hormone GH1 (22 kDa) is a commonly measured biomarker for diagnosis and during treatment of growth disorders, but its quantification by ligand binding assays may be compromised by the occurrence of a number of isoforms. These can interfere in the assays and lead to differences in results between laboratories and potentially even in the treatment of patients. We present an LC-MS/MS method that is able to distinguish the major growth hormone isoform (GH1, 22 kDa) from other isoforms and quantify it without any interference across the clinically relevant concentration range of 0.5 to 50 ng/mL. Analysis involves purification of a 100-µL serum sample by immunocapture using an anti-GH-directed antibody, tryptic digestion, and LC-MS/MS quantification of an isoform-specific signature peptide for GH1 (22 kDa). A tryptic peptide occurring in all GH isoforms is monitored in the same 16-min analytical run as a read-out for total GH. Stable-isotope-labeled forms of these two peptides are included as internal standards. Full validation of the method according to recent guidelines, against a recombinant form of the analyte in rat plasma calibrators, demonstrated intra-assay and inter-assay imprecision below 6% across the calibration range for both signature peptides and recoveries between 94 and 102%. An excellent correlation was found between nominal and measured concentrations of the WHO reference standard for GH1 (22 kDa). Addition of up to 1000 ng/mL biotin or the presence of a 100-fold excess of GH binding protein did not affect the measurement. Equivalent method performance was found for analysis of GH in serum, EDTA, and heparin plasma. Analyte stability was demonstrated during all normal sample storage conditions. Comparison with the IDS-iSYS GH immunoassay showed a good correlation with the LC-MS/MS method for the isoform-specific signature peptide, but a significant positive bias was observed for the LC-MS/MS results of the peptide representing total GH. This seems to confirm the actual occurrence of other GH isoforms in serum. Finally, in serum from pregnant individuals, no quantifiable GH1 (22 kDa) was found, but relatively high concentrations of total GH.

Pipelines and Systems for Threshold-Avoiding Quantification of LC-MS/MS Data.

The accurate processing of complex liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) data from biological samples is a major challenge for metabolomics, proteomics, and related approaches. Here, we present the pipelines and systems for threshold-avoiding quantification (PASTAQ) LC-MS/MS preprocessing toolset, which allows highly accurate quantification of data-dependent acquisition LC-MS/MS datasets. PASTAQ performs compound quantification using single-stage (MS1) data and implements novel algorithms for high-performance and accurate quantification, retention time alignment, feature detection, and linking annotations from multiple identification engines. PASTAQ offers straightforward parameterization and automatic generation of quality control plots for data and preprocessing assessment. This design results in smaller variance when analyzing replicates of proteomes mixed with known ratios and allows the detection of peptides over a larger dynamic concentration range compared to widely used proteomics preprocessing tools. The performance of the pipeline is also demonstrated in a biological human serum dataset for the identification of gender-related proteins.

Creatine homeostasis and protein energy wasting in hemodialysis patients.

Muscle wasting, low protein intake, hypoalbuminemia, low body mass, and chronic fatigue are prevalent in hemodialysis patients. Impaired creatine status may be an often overlooked, potential contributor to these symptoms. However, little is known about creatine homeostasis in hemodialysis patients. We aimed to elucidate creatine homeostasis in hemodialysis patients by assessing intradialytic plasma changes as well as intra- and interdialytic losses of arginine, guanidinoacetate, creatine and creatinine. Additionally, we investigated associations of plasma creatine concentrations with low muscle mass, low protein intake, hypoalbuminemia, low body mass index, and chronic fatigue. Arginine, guanidinoacetate, creatine and creatinine were measured in plasma, dialysate, and urinary samples of 59 hemodialysis patients. Mean age was 65 ± 15 years and 63% were male. During hemodialysis, plasma concentrations of arginine (77 ± 22 to 60 ± 19 µmol/L), guanidinoacetate (1.8 ± 0.6 to 1.0 ± 0.3 µmol/L), creatine (26 [16-41] to 21 [15-30] µmol/L) and creatinine (689 ± 207 to 257 ± 92 µmol/L) decreased (all P < 0.001). During a hemodialysis session, patients lost 1939 ± 871 µmol arginine, 37 ± 20 µmol guanidinoacetate, 719 [399-1070] µmol creatine and 15.5 ± 8.4 mmol creatinine. In sex-adjusted models, lower plasma creatine was associated with a higher odds of low muscle mass (OR per halving: 2.00 [1.05-4.14]; P = 0.04), low protein intake (OR: 2.13 [1.17-4.27]; P = 0.02), hypoalbuminemia (OR: 3.13 [1.46-8.02]; P = 0.008) and severe fatigue (OR: 3.20 [1.52-8.05]; P = 0.006). After adjustment for potential confounders, these associations remained materially unchanged. Creatine is iatrogenically removed during hemodialysis and lower plasma creatine concentrations were associated with higher odds of low muscle mass, low protein intake, hypoalbuminemia, and severe fatigue, indicating a potential role for creatine supplementation.

Diagnostic Accuracy of Salivary Metanephrines in Pheochromocytomas and Paragangliomas.

BACKGROUND: Measurements of plasma free metanephrines are recommended for diagnosing pheochromocytomas and paragangliomas (PPGL). Metanephrines can be detected in saliva with LC-MS/MS with sufficient analytical sensitivity and precision. Because collecting saliva is noninvasive and less cumbersome than plasma or urine sampling, we assessed the diagnostic accuracy of salivary metanephrines in diagnosing PPGL. METHODS: This 2-center study included 118 healthy participants (44 men; mean age: 33 years (range: 19--74 years)), 44 patients with PPGL, and 54 patients suspected of PPGL. Metanephrines were quantified in plasma and saliva using LC-MS/MS. Diagnostic accuracy; correlation between plasma and salivary metanephrines; and potential factors influencing salivary metanephrines, including age, sex, and posture during sampling, were assessed. RESULTS: Salivary metanephrines were significantly higher in patients with PPGL compared with healthy participants (metanephrine (MN): 0.19 vs 0.09 nmol/L, P < 0.001; normetanephrine (NMN): 2.90 vs 0.49 nmol/L, P < 0.001). The diagnostic sensitivity and specificity of salivary metanephrines were 89% and 87%, respectively. Diagnostic accuracy of salivary metanephrines was 88%, with an area under the ROC curve of 0.880. We found a significant correlation between plasma and salivary metanephrines (Pearson correlation coefficient: MN, 0.86, P < 0.001; NMN, 0.83, P < 0.001). Salivary NMN concentrations were higher when collected in a seated position compared with supine (P < 0.001) and increased with age (P < 0.001). CONCLUSIONS: Salivary metanephrines are a promising tool in the biochemical diagnosis of PPGL. Salivary metanephrines correlate with plasma free metanephrines and are increased in patients with PPGL. At this time, however, salivary metanephrines cannot replace measurement of plasma free metanephrines.

Harmonization of LC-MS/MS Measurements of Plasma Free Normetanephrine, Metanephrine, and 3-Methoxytyramine.

BACKGROUND: Plasma-free normetanephrine and metanephrine (metanephrines) are the recommended biomarkers for testing of pheochromocytoma and paraganglioma (PPGL). This study evaluated the status of harmonization of liquid chromatography-tandem mass spectrometry-based measurements of plasma metanephrines and methoxytyramine and clinical interpretation of test results. METHODS: 125 plasma samples from patients tested for PPGLs were analyzed in 12 laboratories. Analytical performance was also assessed from results of a proficiency-testing program. Agreement of test results from different laboratories was assessed by Passing-Bablok regression and Bland-Altman analysis. Agreement in clinical test interpretation based on laboratory specific reference intervals was also examined. RESULTS: Comparisons of analytical test results by regression analysis revealed strong correlations for normetanephrine and metanephrine (R ≥ 0.95) with mean slopes of 1.013 (range 0.975-1.078), and 1.019 (range 0.963-1.081), and intercepts of -0.584 (-53.736 to 54.790) and -3.194 (-17.152 to 5.933), respectively. The mean bias between methods was 1.2% (-11.6% to 16.0%) for metanephrine and 0.1% (-18.0% to 9.5%) for normetanephrine. Measurements of 3-methoxytyramine revealed suboptimal agreement between laboratories with biases ranging from -32.2% to 64.0%. Interrater agreement in test interpretation was >94% for metanephrine and >84% for normetanephrine; improvements in interrater agreement were observed with use of harmonized reference intervals, including age-specific cut-offs for normetanephrine. CONCLUSIONS: Analytical methods for metanephrines are well harmonized between laboratories. However, the 16% disagreement in test interpretation for normetanephrine suggests use of suboptimal method-dependent reference intervals for clinical decision-making for this metabolite. Improved analytical methods and reference interval harmonization are particularly required for 3-methoxytyramine.

Correlations of blood and brain biochemistry in phenylketonuria: Results from the Pah-enu2 PKU mouse.

BACKGROUND: In phenylketonuria (PKU), treatment monitoring is based on frequent blood phenylalanine (Phe) measurements, as this is the predictor of neurocognitive and behavioural outcome by reflecting brain Phe concentrations and brain biochemical changes. Despite clinical studies describing the relevance of blood Phe to outcome in PKU patients, blood Phe does not explain the variance in neurocognitive and behavioural outcome completely. METHODS: In a PKU mouse model we investigated 1) the relationship between plasma Phe and brain biochemistry (Brain Phe and monoaminergic neurotransmitter concentrations), and 2) whether blood non-Phe Large Neutral Amino Acids (LNAA) would be of additional value to blood Phe concentrations to explain brain biochemistry. To this purpose, we assessed blood amino acid concentrations and brain Phe as well as monoaminergic neurotransmitter levels in in 114 Pah-Enu2 mice on both B6 and BTBR backgrounds using (multiple) linear regression analyses. RESULTS: Plasma Phe concentrations were strongly correlated to brain Phe concentrations, significantly negatively correlated to brain serotonin and norepinephrine concentrations and only weakly correlated to brain dopamine concentrations. From all blood markers, Phe showed the strongest correlation to brain biochemistry in PKU mice. Including non-Phe LNAA concentrations to the multiple regression model, in addition to plasma Phe, did not help explain brain biochemistry. CONCLUSION: This study showed that blood Phe is still the best amino acid predictor of brain biochemistry in PKU. Nevertheless, neurocognitive and behavioural outcome cannot fully be explained by blood or brain Phe concentrations, necessitating a search for other additional parameters. TAKE-HOME MESSAGE: Blood Phe is still the best amino acid predictor of brain biochemistry in PKU. Nevertheless, neurocognitive and behavioural outcome cannot fully be explained by blood or brain Phe concentrations, necessitating a search for other additional parameters.

Plasma creatine and incident type 2 diabetes in a general population-based cohort: The PREVEND study.

BACKGROUND: Type 2 diabetes is associated with both impaired insulin action at target tissues and impaired insulin secretion in pancreatic beta cells. Mitochondrial dysfunction may play a role in both insulin resistance and impaired insulin secretion. Plasma creatine has been proposed as a potential marker for mitochondrial dysfunction. We aimed to investigate the association between plasma creatine and incident type 2 diabetes. METHODS: We measured fasting plasma creatine concentrations by nuclear magnetic resonance spectroscopy in participants of the general population-based PREVEND study. The study outcome was incident type 2 diabetes, defined as a fasting plasma glucose ≥7.0 mmol/L (126 mg/dl); a random sample plasma glucose ≥11.1 mmol/L (200 mg/dl); self-report of a physician diagnosis or the use of glucose-lowering medications based on a central pharmacy registration. Associations of plasma creatine with type 2 diabetes were quantified using Cox proportional hazards models and were adjusted for potential confounders. RESULTS: We included 4735 participants aged 52 ± 11 years, of whom 49% were male. Mean plasma creatine concentrations were 36.7 ± 17.6 µmol/L, with lower concentrations in males than in females (30.4 ± 15.1 µmol/L vs. 42.7 ± 17.7 µmol/L; p for difference <.001). During 7.3 [6.2-7.7] years of follow-up, 235 (5.4%) participants developed type 2 diabetes. Higher plasma creatine concentrations were associated with an increased risk of incident type 2 diabetes (HR per SD change: 1.27 [95% CI: 1.11-1.44]; p < .001), independent of potential confounders. This association was strongly modified by sex (p interaction <.001). Higher plasma creatine was associated with an increased risk of incident type 2 diabetes in males (HR: 1.40 [1.17-1.67]; p < .001), but not in females (HR: 1.10 [0.90-1.34]; p = .37). CONCLUSION: Fasting plasma creatine concentrations are lower in males than in females. Higher plasma creatine is associated with an increased risk of type 2 diabetes in males.

The novel TRPA1 antagonist BI01305834 inhibits ovalbumin-induced bronchoconstriction in guinea pigs.

BACKGROUND: Asthma is a chronic respiratory disease in which the nervous system plays a central role. Sensory nerve activation, amongst others via Transient Receptor Potential Ankyrin 1 (TRPA1) channels, contributes to asthma characteristics including cough, bronchoconstriction, mucus secretion, airway hyperresponsiveness (AHR) and inflammation. In the current study, we evaluated the efficacy of the novel TRPA1 antagonist BI01305834 against AHR and inflammation in guinea-pig models of asthma. METHODS: First, a pilot study was performed in a guinea-pig model of allergic asthma to find the optimal dose of BI01305834. Next, the effect of BI01305834 on (1) AHR to inhaled histamine after the early and late asthmatic reaction (EAR and LAR), (2) magnitude of EAR and LAR and (3) airway inflammation was assessed. Precision-cut lung slices and trachea strips were used to investigate the bronchoprotective and bronchodilating-effect of BI01305834. Statistical evaluation of differences of in vivo data was performed using a Mann-Whitney U test or One-way nonparametric Kruskal-Wallis ANOVA, for ex vivo data One- or Two-way ANOVA was used, all with Dunnett's post-hoc test where appropriate. RESULTS: A dose of 1 mg/kg BI01305834 was selected based on AHR and exposure data in blood samples from the pilot study. In the subsequent study, 1 mg/kg BI01305834 inhibited AHR after the EAR, and the development of EAR and LAR elicited by ovalbumin in ovalbumin-sensitized guinea pigs. BI01305834 did not inhibit allergen-induced total and differential cells in the lavage fluid and interleukin-13 gene expression in lung homogenates. Furthermore, BI01305834 was able to inhibit allergen and histamine-induced airway narrowing in guinea-pig lung slices, without affecting histamine release, and reverse allergen-induced bronchoconstriction in guinea-pig trachea strips. CONCLUSIONS: TRPA1 inhibition protects against AHR and the EAR and LAR in vivo and allergen and histamine-induced airway narrowing ex vivo, and reverses allergen-induced bronchoconstriction independently of inflammation. This effect was partially dependent upon histamine, suggesting a neuronal and possible non-neuronal role for TRPA1 in allergen-induced bronchoconstriction.

Clinical presentation and long-term follow-up of dopamine beta hydroxylase deficiency.

Dopamine beta hydroxylase (DBH) deficiency is an extremely rare autosomal recessive disorder with severe orthostatic hypotension, that can be treated with L-threo-3,4-dihydroxyphenylserine (L-DOPS). We aimed to summarize clinical, biochemical, and genetic data of all world-wide reported patients with DBH-deficiency, and to present detailed new data on long-term follow-up of a relatively large Dutch cohort. We retrospectively describe 10 patients from a Dutch cohort and 15 additional patients from the literature. We identified 25 patients (15 females) from 20 families. Ten patients were diagnosed in the Netherlands. Duration of follow-up of Dutch patients ranged from 1 to 21 years (median 13 years). All patients had severe orthostatic hypotension. Severely decreased or absent (nor)epinephrine, and increased dopamine plasma concentrations were found in 24/25 patients. Impaired kidney function and anemia were present in all Dutch patients, hypomagnesaemia in 5 out of 10. Clinically, all patients responded very well to L-DOPS, with marked reduction of orthostatic complaints. However, orthostatic hypotension remained present, and kidney function, anemia, and hypomagnesaemia only partially improved. Plasma norepinephrine increased and became detectable, while epinephrine remained undetectable in most patients. We confirm the core clinical characteristics of DBH-deficiency and the pathognomonic profile of catecholamines in body fluids. Impaired renal function, anemia, and hypomagnesaemia can be part of the clinical presentation. The subjective response to L-DOPS treatment is excellent and sustained, although the neurotransmitter profile in plasma does not normalize completely. Furthermore, orthostatic hypotension as well as renal function, anemia, and hypomagnesaemia improve only partially.

An antibody-free LC-MS/MS method for the quantification of intact insulin-like growth factors 1 and 2 in human plasma.

Insulin-like growth factors 1 and 2 (IGF-1 and IGF-2) are important biomarkers in research and diagnosis of growth disorders. Quantitative analysis is performed using various ligand-binding assays or enzymatic digestion LC-MS/MS methods, whose widespread adoption is hampered by time-consuming sample preparation procedures. We present a simple and fast antibody-free LC-MS/MS method for the quantification of intact IGF-1 and IGF-2 in human plasma. The method requires 50 µL of plasma and uses fully 15N-labelled IGF-1 as internal standard. It features trifluoroethanol (TFE)-based IGF/IGF-binding protein complex dissociation and a two-step selective protein precipitation workflow, using 5% acetic acid in 80/20 acetone/acetonitrile (precipitation 1) and ice-cold ethanol (precipitation 2). Detection of intact IGF-1 and IGF-2 is performed by means of a Waters XEVO TQ-S triple quadrupole mass spectrometer in positive electrospray ionisation (ESI+) mode. Lower limits of quantification were 5.9 ng/mL for IGF-1 and 8.4 ng/mL for IGF-2. Intra-assay imprecision was below 4.5% and inter-assay imprecision was below 5.8% for both analytes. An excellent correlation was found between nominal and measured concentrations of the WHO reference standard for IGF-1. Comparison with the IDS-iSYS IGF-1 immunoassay showed good correlation (R2 > 0.97), although a significant bias was observed with the immunoassay giving substantially higher concentrations. The LC-MS/MS method described here allows for reliable and simultaneous quantification of IGF-1 and IGF-2 in plasma, without the need for enzymatic digestion. The method can be readily implemented in clinical mass spectrometry laboratories and has the potential to be adapted for the analysis of different similarly sized peptide hormones.

Combined low vitamin D and K status amplifies mortality risk: a prospective study.

OBJECTIVE: To explore the association of both plasma vitamin D and K concentrations with all-cause mortality, cardiovascular mortality, and cardiovascular events in the general population. METHODS: We studied 4742 participants of the Prevention of REnal and Vascular ENd-Stage Disease (PREVEND) Study. At baseline, vitamin D and K status was determined by measurement of 25-hydroxyvitamin D [25(OH)D] and dephosphorylated uncarboxylated matrix Gla protein (dp-ucMGP), respectively. Patients were categorized into: 25(OH)D < 50 or ≥ 50 nmol/L and dp-ucMGP < 361 or ≥ 361 pmol/L with 25(OH)D > 75 nmol/L and dp-ucMGP < 361 pmol/L as reference. Cause of death was coded according to International Classification of Diseases 9&10 codes from the 2001-2003 examination until date of death/event or censoring date (January 1st, 2017). RESULTS: Mean age was 52.6 ± 11.9 years and 2513 (53%) were female. During a median of 14.2 year follow-up, 620 participants died of which 142 were due to cardiovascular causes. Combined low vitamin D and K status was present in 970 participants (20%) and was associated with a greater risk of all-cause mortality compared to high vitamin D and high vitamin K status group (n = 1424) after adjusting for potential confounders: hazard ratio 1.46 (95% confidence intervals 1.12-1.90). We observed similar trends, albeit non-significant for cardiovascular mortality, and cardiovascular events: 1.42 (0.79-2.55), 1.28 (0.93-1.77), respectively. CONCLUSIONS: Combined low vitamin D and K status are associated with increased all-cause mortality risk and possibly with cardiovascular mortality and cardiovascular events compared with adequate vitamin D and K status. Future studies should investigate the effect of combined vitamin D and K supplementation on clinical outcomes.