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.

Associations of 24 h urinary excretions of α- and γ-carboxyethyl hydroxychroman with plasma α- and γ-tocopherol and dietary vitamin E intake in older adults: the Lifelines-MINUTHE Study.

BACKGROUND: Urinary metabolites of vitamin E, i.e., α- and γ-carboxyethyl hydroxychroman (α- and γ-CEHC), have gained increasing attention and have been proposed as novel biomarkers of vitamin E intake and status. However, there are insufficient data on the relationship of plasma α-tocopherol and γ-tocopherol and dietary vitamin E intake with 24 h urinary excretions of α- and γ-CEHC. OBJECTIVES: We aimed to (1) investigate the associations of urinary α- and γ-CEHC/creatinine ratios and 24 h urinary excretions of α- and γ-CEHC with plasma α- and γ-tocopherol, respectively; (2) investigate the associations of urinary α- and γ-CEHC/creatinine ratios and 24 h urinary excretions of α- and γ-CEHC with dietary vitamin E intake, and we hypothesize that 24 h urinary excretions of α- and γ-CEHC will better correlate with vitamin E intake than urinary α- and γ-CEHC/creatinine ratios. DESIGN: 24 h Urine and plasma samples were collected from 1519 participants (60-75 years, male: 50%) included in the Lifelines-MINUTHE Study for the assessments of urinary α- and γ-CEHC/creatinine ratios and 24 h urinary excretions of α- and γ-CEHC, and plasma α- and γ-tocopherol. Among those participants, dietary vitamin E intake data from 387 participants were available from an externally validated Flower-Food Frequency Questionnaire (FFQ). The associations of plasma α- and γ-tocopherol, dietary vitamin E intake, with urinary α- and γ-CEHC were assessed using multivariate linear regressions. RESULTS: 24 h Urinary excretion of α-CEHC (median (IQR): 0.9 (0.3-2.4) µmol) was less than that of γ-CEHC (median (IQR): 1.5 (0.5-3.5) µmol). After adjustment for covariates, we found that 24 h urinary α-CEHC excretion and urinary α-CEHC/creatinine ratio were both positively associated with plasma α-tocopherol (std.beta: 0.06, p = 0.02; std.beta: 0.06, p = 0.01, respectively). Furthermore, the sum of 24 h urinary α- and γ-CEHC excretions was positively associated with dietary vitamin E intake (std.beta: 0.08; p = 0.03), whereas there was no relation between urinary α- and γ-CEHC/creatinine ratios and vitamin E intake. No association was observed neither between plasma α- and γ-tocopherol and dietary vitamin E intake, nor between urinary γ-CEHC and plasma γ-tocopherol. CONCLUSION: Our study confirmed our hypothesis that 24 h urinary α- and γ-CEHC excretions would be a better marker for dietary vitamin E intake than urinary α- and γ-CEHC/creatinine ratios. Considering that both 24 h urinary α- and γ-CEHC excretions and α- and γ-CEHC/creatinine ratios were also associated with plasma α-tocopherol status, we suggest that 24 h urinary α- and γ-CEHC excretions could be used to assess overall vitamin E status.

Long-term magnesium supplementation improves glucocorticoid metabolism: A post-hoc analysis of an intervention trial.

OBJECTIVE: Increasing magnesium intake might reduce the risk of cardiovascular disease (CVD). Whether potential effects on cortisol contribute to these beneficial effects on cardiovascular health remains unclear. We therefore studied effects of long-term oral magnesium supplementation on glucocorticoid metabolism, specifically on the excretion of urinary cortisol, cortisone and their metabolites, as well as on the ratios reflecting enzymatic activity of 11ß-hydroxysteroid dehydrogenases (11ß-HSDs) and A-ring reductases. DESIGN: A post-hoc analysis of a randomized trial with allocation to a magnesium supplement (350 mg/day) or a placebo for 24-week. PATIENTS: Forty-nine overweight men and women, aged between 45 and 70 years. MEASUREMENTS: Cortisol, cortisone and their metabolites (tetrahydrocortisol [THF], allo-tetrahydrocortisol [allo-THF] and tetrahydrocortisone [THE]) were measured in 24-h urine samples. Enzymatic activities of 11ß-HSD overall and of 11ß-HSD type 2 were estimated as the urinary (THF + allo-THF [THFs])/THE and cortisol/cortisone ratios, respectively. A-ring reductase activity was assessed by ratios of THF/allo-THF, allo-THF/cortisol, THF/cortisol and THE/cortisone. RESULTS: After 24-week, urinary cortisol excretion was decreased in the magnesium group as compared with the placebo group (-32 nmol/24-h, 95% CI: -59; -5 nmol/24-h, p = .021). Ratios of THFs/THE and cortisol/cortisone were decreased following magnesium supplementation by 0.09 (95% CI: 0.02; 0.17, p = .018) and 0.10 (95% CI: 0.03; 0.17, p = .005), respectively. No effects were observed on A-ring reductase activity. CONCLUSIONS: We observed a beneficial effect of magnesium supplementation towards a lower 24-h urinary cortisol excretion together with an increased activity of 11ß-HSD type 2. Our findings may provide another potential mechanism by which increased magnesium intake lowers CVD risk (ClinicalTrials.gov identifier: NCT02235805).

Methylmalonic acid, vitamin B12, renal function, and risk of all-cause mortality in the general population: results from the prospective Lifelines-MINUTHE study.

BACKGROUND: Methylmalonic acid (MMA) is best known for its use as a functional marker of vitamin B12 deficiency. However, MMA concentrations not only depend on adequate vitamin B12 status, but also relate to renal function and endogenous production of propionic acid. Hence, we aimed to investigate to what extent variation in MMA levels is explained by vitamin B12 and eGFR and whether MMA levels are associated with mortality if vitamin B12 and eGFR are taken into account. METHODS: A total of 1533 individuals (aged 60-75 years, 50% male) were included from the Lifelines Cohort and Biobank Study. Individuals were included between 2006 and 2013, and the total follow-up time was 8.5 years. RESULTS: Median [IQR] age of the study population was 65 [62-69] years, 50% was male. At baseline, median MMA concentration was 170 [138-216] nmol/L, vitamin B12 290 [224-362] pmol/L, and eGFR 84 [74-91] mL/min/1.73 m2. Log2 vitamin B12, log2 eGFR, age, and sex were significantly associated with log2 MMA in multivariable linear regression analyses (model R2 = 0.22). After a total follow-up time of 8.5 years, 72 individuals had died. Log2 MMA levels were significantly associated with mortality (hazard ratio [HR] 1.67 [95% CI 1.25-2.22], P < 0.001). Moreover, we found a significant interaction between MMA and eGFR with respect to mortality (Pinteraction < 0.001). CONCLUSIONS: Only 22% of variation in MMA levels was explained by vitamin B12, eGFR, age, and sex, indicating that a large part of variation in MMA levels is attributable to other factors (e.g., catabolism, dietary components, or gut microbial production). Higher MMA levels are associated with an increased risk for mortality, independent of vitamin B12, eGFR, and sex. This association was more pronounced in individuals with impaired renal function.

Effect of Vasopressin on the Hypothalamic-Pituitary-Adrenal Axis in ADPKD Patients during V2 Receptor Antagonism.

BACKGROUND: Patients with autosomal dominant polycystic kidney disease (ADPKD) are treated with a vasopressin V2 receptor antagonist (V2RA) to slow disease progression. This drug increases vasopressin considerably in these patients with already elevated baseline levels. Vasopressin is known to stimulate the hypothalamic-pituitary-adrenal (HPA) axis through V1 and V3 receptor activation. It is unknown whether this increase in vasopressin during V2RA treatment affects glucocorticoid production. METHODS: Twenty-seven ADPKD patients were studied on and off treatment with a V2RA and compared to age- and sex-matched healthy controls and IgA nephropathy patients, the latter also matched for kidney function. Vasopressin was measured by its surrogate copeptin. Twenty-four-hour urinary excretions of cortisol, cortisone, tetrahydrocortisone, tetrahydrocortisol, allotetrahydrocortisol, and the total glucocorticoid pool were measured. RESULTS: At baseline, ADPKD patients demonstrated a higher copeptin concentration in comparison with healthy controls, while urinary excretion of cortisol and cortisone was lower (medians of 0.23 vs. 0.34 µmol/24 h, p = 0.007, and 0.29 vs. 0.53 µmol/24 h, p < 0.001, respectively). There were no differences in cortisol and cortisone excretion compared to IgA nephropathy patients. Cortisol, cortisone, and total glucocorticoid excretions correlated with kidney function (R = 0.37, 0.58, and 0.19, respectively; all p < 0.05). Despite that V2RA treatment resulted in a 3-fold increase in copeptin, only cortisone excretion increased (median of 0.44 vs. baseline 0.29 µmol/24 h, p < 0.001), whereas no changes in cortisol or total glucocorticoid excretion were observed. CONCLUSIONS: Increased concentration of vasopressin in ADPKD patients at baseline and during V2RA treatment does not result in activation of the HPA axis. The impaired glucocorticoid production in these patients is related to their degree of kidney function impairment.

Endogenous urinary glucocorticoid metabolites and mortality in prednisolone-treated renal transplant recipients.

BACKGROUND: Chronic corticosteroid treatment suppresses HPA-axis activity and might alter activity of 11ß hydroxysteroid dehydrogenases (11ß-HSD). We aimed to investigate whether the endogenous glucocorticoid production and 11ß-HSD activities are altered in prednisolone-treated renal transplant recipients (RTR) compared with healthy controls and whether this has implications for long-term survival in RTR. METHODS: In a longitudinal cohort of 693 stable RTR and 275 healthy controls, 24-hour urinary cortisol, cortisone, tetrahydrocorisol (THF), allotetrahydrocortisol (alloTHF), and tetrahydrocortisone (THE) were measured using liquid chromatography tandem-mass spectrometry. Twenty-four-hour urinary excretion of cortisol and metabolites were used as measures of endogenous glucocorticoid production; (THF + alloTHF)/THE and cortisol/cortisone ratios were used as measures of 11ß-HSD activity. RESULTS: Urinary cortisol and metabolite excretion were significantly lower in RTR compared with healthy controls (P < .001), whereas (THF + alloTHF)/THE and cortisol/cortisone ratios were significantly higher (P < .001 and P = .002). Lower total urinary metabolite excretion and higher urinary (THF + alloTHF)/THE ratios were associated with increased risk of mortality, independent of age, sex, estimated glomerular filtration rate, C-reactive protein, body surface area, and daily prednisolone dose, respectively. CONCLUSIONS: Endogenous glucocorticoid production and 11ß-HSD activities are altered in prednisolone-treated RTR. Decreased total urinary endogenous glucocorticoid metabolite excretion and increased urinary (THF + alloTHF)/THE ratios are associated with increased risk of mortality.

Urinary sulfate excretion and risk of late graft failure in renal transplant recipients - a prospective cohort study.

Hydrogen sulfide (H2 S), produced from metabolism of dietary sulfur-containing amino acids, is allegedly a renoprotective compound. Twenty-four-hour urinary sulfate excretion (USE) may reflect H2 S bioavailability. We aimed to investigate the association of USE with graft failure in a large prospective cohort of renal transplant recipients (RTR). We included 704 stable RTR, recruited at least 1 year after transplantation. We applied log-rank testing and Cox regression analyses to study association of USE, measured from baseline 24 h urine samples, with graft failure. Median age was 55 [45-63] years (57% male, eGFR was 45 ± 19 ml/min/1.73 m2 ). Median USE was 17.1 [13.1-21.1] mmol/24 h. Over median follow-up of 5.3 [4.5-6.0] years, 84 RTR experienced graft failure. RTR in the lowest sex-specific tertile of USE experienced a higher rate of graft failure during follow-up than RTR in the middle and highest sex-specific tertiles (18%, 13%, and 5%, respectively, log-rank P < 0.001). In Cox regression analyses, USE was inversely associated with graft failure [HR per 10 mmol/24 h: 0.37 (0.24-0.55), P < 0.001]. The association remained independent of adjustment for potential confounders, including age, sex, eGFR, proteinuria, time between transplantation and baseline, BMI, smoking, and high sensitivity C-reactive protein [HR per 10 mmol/24 h: 0.51 (0.31-0.82), P = 0.01]. In conclusion, this study demonstrates a significant inverse association of USE with graft failure in RTR, suggesting high H2 S bioavailability as a novel, potentially modifiable factor for prevention of graft failure in RTR.

High plasma guanidinoacetate-to-homoarginine ratio is associated with high all-cause and cardiovascular mortality rate in adult renal transplant recipients.

L-Arginine:glycine amidinotransferase (AGAT) is the main producer of the creatine precursor, guanidinoacetate (GAA), and L-homoarginine (hArg). We and others previously reported lower levels of circulating and urinary hArg in renal transplant recipients (RTR) compared to healthy subjects. In adults, hArg emerged as a novel risk factor for renal and cardiovascular adverse outcome. Urinary GAA was found to be lower in children and adolescents with kidney transplants compared to healthy controls. Whether GAA is also a risk factor in the renal and cardiovascular systems of adults, is not yet known. In the present study, we aimed to investigate the significance of circulating GAA and the GAA-to-hArg molar ratio (GAA/hArg) in adult RTR. We hypothesized that GAA/hArg represents a measure of the balanced state of the AGAT activity in the kidneys, and would prospectively allow assessing a potential association between GAA/hArg and long-term outcome in RTR. The median follow-up period was 5.4 years. Confounders and potential mediators of GAA/hArg associations were evaluated with multivariate linear regression analyses, and the association with all-cause and cardiovascular mortality or death-censored graft loss was studied with Cox regression analyses. The study cohort consisted of 686 stable RTR and 140 healthy kidney donors. Median plasma GAA concentration was significantly lower in the RTR compared to the kidney donors before kidney donation: 2.19 [1.77-2.70] µM vs. 2.78 [2.89-3.35] µM (P < 0.001). In cross-sectional multivariable analyses in RTR, HDL cholesterol showed the strongest association with GAA/hArg. In prospective analyses in RTR, GAA/hArg was associated with a higher risk for all-cause mortality (hazard ratio (HR): 1.35 [95% CI 1.19-1.53]) and cardiovascular mortality (HR: 1.46 [95% CI 1.24-1.73]), independent of potential confounders. GAA but not GAA/hArg was associated with death-censored graft loss in crude survival and Cox regression analyses. The association of GAA and death-censored graft loss was lost after adjustment for eGFR. Our study suggests that in the kidneys of RTR, the AGAT-catalyzed biosynthesis of GAA is decreased. That high GAA/hArg is associated with a higher risk for all-cause and cardiovascular mortality may suggest that low plasma hArg is a stronger contributor to these adverse outcomes in RTR than GAA.

Plasma ADMA, urinary ADMA excretion, and late mortality in renal transplant recipients.

Cardiovascular disease (CVD) is the leading cause of death in renal transplant recipients (RTR). Elevated plasma asymmetric dimethylarginine (pADMA), an endogenous nitric oxide synthase inhibitor produced from the turnover of methylated arginine moieties in proteins, is a risk factor for CVD and mortality. It is unknown how urinary ADMA excretion (uADMA), one of the main ADMA elimination routes, is associated with long-term survival. Furthermore, the association of pADMA and uADMA with markers for turnover of arginine-methylated proteins is unknown. We analyzed ADMA using a validated GC-MS/MS method in plasma and 24-h urine samples of 685 RTR, included ≥ 1 year after transplantation. We also analyzed urine symmetric dimethylarginine (uSDMA) using the same method. Urinary creatinine and urea excretions were used as markers for turnover of muscle protein and amino acids, respectively. We applied Cox regression analyses to study associations of pADMA, uADMA, and uSDMA with all-cause and CVD mortality. Mean pADMA was 0.61 ± 0.12 µM, uADMA was 31 ± 13 µmol/24 h, and uSDMA was 52 ± 19 µmol/24 h. Over median follow-up of 5.4 [4.9-6.1] years, 147 RTR died, of which 58 (39%) from CVD. High pADMA was associated with high all-cause mortality (HR per SD [95% CI]: 1.45 [1.26-1.67], P < 0.001), while high uADMA was associated with low all-cause and CVD mortality (HR per SD [95% CI]: 0.57 [0.47-0.69], P < 0.001, and 0.55 [0.40-0.74], P < 0.001, respectively). The associations were independent of adjustment for potential confounders. Creatinine excretion was associated with both pADMA (st. ß:- 0.21, P = 0.003) and uADMA (st. ß: 0.49, P < 0.001), and urea excretion was associated with uADMA (st. ß: 0.56, P < 0.001). Associations of uSDMA with outcomes and with creatinine excretion and urea excretion were comparable to those of uADMA. The associations of pADMA, uADMA and uSDMA with mortality were strongly affected by adjustment for creatinine excretion and urea excretion. We found for the first time that high uADMA and high uSDMA are associated with less risk of all-cause and CVD mortality. The links of uADMA and uSDMA with markers of muscle protein and amino acid turnover may serve to further understand ADMA and SDMA homeostasis and their clinical implications.

Effect of renal function on homeostasis of asymmetric dimethylarginine (ADMA): studies in donors and recipients of renal transplants.

Asymmetric dimethylarginine (ADMA) is a methylated form of arginine and an endogenous nitric oxide synthase inhibitor. Renal function decline is associated with increase of plasma ADMA in chronic kidney disease populations. It is yet unknown how isolated renal function impairment affects ADMA homeostasis in healthy humans. Here, we measured plasma concentrations and urinary excretion of ADMA using GC-MS/MS in 130 living kidney donors before and at 1.6 (1.6-1.9) months after donation. We additionally analyzed 201 stable renal transplant recipients (RTR) that were included > 1 year after transplantation, as a model for kidney disease in the context of single kidney state. We measured true glomerular filtration rate (mGFR) using 125I-iothalamate. To study enzymatic metabolism of ADMA, we also measured L-citrulline as primary metabolite. Mean age was 52 ± 10 years in donors and 54 ± 12 years in RTR. Renal function was significantly reduced from pre- to post-donation (mGFR: 104 ± 17 vs. 66 ± 10 ml/min per 1.73 m2 BSA, - 36 ± 7%, P < 0.001). Urinary ADMA excretion strongly and significantly decreased from pre- to post-donation (60.6 ± 16.0 vs. 40.5 ± 11.5 µmol/24 h, - 31.5 ± 21.5%, P < 0.001), while plasma ADMA increased only slightly (0.53 ± 0.08 vs. 0.58 ± 0.09 µM, 11.1 ± 20.1%, P < 0.001). Compared to donors post-donation, RTR had significantly worse renal function (mGFR: 49 ± 18 ml/min/1.73 m2, - 25 ± 2%, P < 0.001) and lower urinary ADMA excretion (30.9 ± 12.4 µmol/24 h, - 23.9 ± 3.4%, P < 0.001). Plasma ADMA in RTR (0.60 ± 0.11 µM) did not significantly differ from donors post-donation (2.9 ± 1.9%, P = 0.13). Plasma citrulline was inversely associated with mGFR (st. ß: - 0.23, P < 0.001), consistent with increased ADMA metabolism to citrulline with lower GFR. In both groups, the response of urinary ADMA excretion to renal function loss was much larger than that of plasma ADMA. As citrulline was associated with GFR, our data indicate that with renal function impairment, a decrease in urinary ADMA excretion does not lead to a corresponding increase in plasma ADMA, likely due to enhanced metabolism, thus allowing for lower renal excretion of ADMA.

The tryptophan/kynurenine pathway, systemic inflammation, and long-term outcome after kidney transplantation.

Tryptophan is metabolized along the kynurenine pathway, initially to kynurenine, and subsequently to cytotoxic 3-hydroxykynurenine. There is increasing interest in this pathway because of its proinflammatory nature, and drugs interfering in it have received increasing attention. We aimed to investigate whether serum and urinary parameters of the tryptophan/kynurenine pathway, and particularly cytotoxic 3-hydroxykynurenine, are associated with systemic inflammation and long-term outcome in renal transplant recipients (RTR). Data were collected in outpatient RTR with a functioning graft for >1 yr. Tryptophan, kynurenine, and 3-hydroxykynurenine in serum and urine were measured using LC-MS/MS. A total of 561 RTR (age: 51 ± 12 yr; 56% male) were included at a median of 6.0 (2.6-11.6) yr posttransplantation. Baseline median serum tryptophan was 40.0 (34.5-46.0) µmol/l, serum kynurenine was 1.8 (1.4-2.2) µmol/l, and serum 3-hydroxykynurenine was 42.2 (31.0-61.7) nmol/l. Serum kynurenine and 3-hydroxykynurenine were strongly associated with parameters of systemic inflammation. During follow-up for 7.0 (6.2-7.5) yr, 51 RTR (9%) developed graft failure and 120 RTR (21%) died. Both serum kynurenine and 3-hydroxykynurenine were independently associated with graft failure [HR 1.72 (1.23-2.41), P = 0.002; and HR 2.03 (1.42-2.90), P < 0.001]. Serum 3-hydroxykynurenine was also independently associated with mortality [HR 1.37 (1.08-1.73), P = 0.01], whereas serum kynurenine was not. Urinary tryptophan/kynurenine pathway parameters were not associated with outcome. Of tryptophan metabolites, serum 3-hydroxykynurenine is cross-sectionally most strongly and consistently associated with systemic inflammation and prospectively with adverse long-term outcome after kidney transplantation. Serum 3-hydroxykynurenine may be an interesting biomarker and target for the evaluation of drugs interfering in the tryptophan/kynurenine pathway.

The fate of sulfate in chronic heart failure.

New leads to advance our understanding of heart failure (HF) pathophysiology are urgently needed. Previous studies have linked urinary sulfate excretion to a favorable cardiovascular risk profile. Sulfate is not only the end product of hydrogen sulfide metabolism but is also directly involved in various (patho)physiological processes, provoking scientific interest in its renal handling. This study investigates sulfate clearance in chronic HF (CHF) patients and healthy individuals and considers its relationship with disease outcome. Parameters related to renal sulfate handling were determined in and compared between 96 previously characterized CHF patients and sex-matched healthy individuals. Among patients, sulfate clearance was analyzed for associations with clinical and outcome parameters. In CHF patients, plasma sulfate concentrations are significantly higher, whereas 24-h urinary excretion, fractional excretion, and clearance of sulfate are significantly lower, compared with healthy individuals. Among patients, sulfate clearance is independently associated with diuretics use, creatinine clearance and 24-h urinary sodium excretion. Sulfate clearance is associated with favorable disease outcome [hazard ratio per SD increase 0.38 (95% confidence interval 0.23-0.63), P < 0.001]. Although significance was lost after adjustment for creatinine clearance, the decrease of sulfate clearance in patients is independent of this parameter, indicating that sulfate clearance is not merely a reflection of renal function. This exploratory study reveals aberrant sulfate clearance as a potential contributor to CHF pathophysiology, with reduced levels in patients and a positive association with favorable disease outcome. Further research is needed to unravel the nature of its involvement and to determine its potential as a biomarker and target for therapy.NEW & NOTEWORTHY Sulfate clearance is decreased in chronic heart failure patients compared with healthy individuals. Among patients, sulfate clearance is positively associated with favorable disease outcome, i.e., a decreased rehospitalization rate and increased patient survival. Hence, decreased sulfate clearance may be involved in the pathophysiology of heart failure.

Low plasma homoarginine concentration is associated with high rates of all-cause mortality in renal transplant recipients.

In renal transplant recipients (RTR), we recently found that low urinary excretion of homoarginine (hArg) is associated with mortality and graft failure. However, it is not known whether such prospective associations also hold true for plasma concentrations of hArg. In the present study, we therefore determined plasma concentrations of hArg in the same cohort, i.e. in 687 RTR (functioning graft ≥1 year), and in 140 healthy donors, before and after kidney donation. Plasma hArg concentrations were significantly lower in RTR compared to healthy controls [1.24 (0.95-1.63) µM vs. 1.58 (1.31-2.03) µM, P < 0.001], and kidney donation resulted in a decrease in plasma hArg concentration to 1.41 (1.10-1.81) µM (P < 0.001). In RTR, multivariable linear regression analysis revealed BMI (ß = 0.124), heart rate (ß = -0.091), pre-emptive transplantation (ß = 0.078), antidiabetic medication (ß = -0.091), eGFR (ß = 0.272), plasma PTH (ß = -0.098), uric acid (ß = 0.137), alkaline phosphatase (ß = -0.100), HDL (ß = -0.111), NT-pro-BNP (ß = -0.166), and urinary urea excretion (ß = 0.139) as main determinants of plasma hArg (all P < 0.05). In RTR, plasma hArg concentration was inversely associated with all-cause [hazard ratio (HR) 0.59 (95% CI 0.50-0.70), P < 0.001] and cardiovascular mortality [HR 0.50 (0.39-0.66), P < 0.001], both expressed per standard deviation change in log-transformed hArg, independent of potential confounders. To conclude, our results suggest that the kidney is a major hArg production site and an important modulator of hArg homeostasis in the renal and cardiovascular systems. Moreover, low plasma hArg is independently associated with increased risk of cardiovascular mortality in RTR, which corroborates the cardiovascular importance of preserving kidney function after transplantation.

Iron deficiency, anemia, and mortality in renal transplant recipients.

Anemia, iron deficiency anemia (IDA), and iron deficiency (ID) are highly prevalent in renal transplant recipients (RTR). Anemia is associated with poor outcome, but the role of ID is unknown. Therefore, we aimed to investigate the association of ID, irrespective of anemia, with all-cause mortality in RTR. Cox regression analyses were used to investigate prospective associations. In 700 RTR, prevalences of anemia, IDA, and ID were 34%, 13%, and 30%, respectively. During follow-up for 3.1 (2.7-3.9) years, 81 (12%) RTR died. In univariable analysis, anemia [HR, 1.72 (95%CI: 1.11-2.66), P = 0.02], IDA [2.44 (1.48-4.01), P < 0.001], and ID [2.04 (1.31-3.16), P = 0.001] were all associated with all-cause mortality. In multivariable analysis, the association of anemia with mortality became weaker after adjustment for ID [1.52 (0.97-2.39), P = 0.07] and disappeared after adjustment for proteinuria and eGFR [1.09 (0.67-1.78), P = 0.73]. The association of IDA with mortality attenuated after adjustment for potential confounders. In contrast, the association of ID with mortality remained independent of potential confounders, including anemia [1.77 (1.13-2.78), P = 0.01]. In conclusion, ID is highly prevalent among RTR and is associated with an increased risk of mortality, independent of anemia. As ID is a modifiable factor, correction of ID could be a target to improve survival.

Separate and combined effects of individual and neighbourhood socio-economic disadvantage on health-related lifestyle risk factors: a multilevel analysis.

BACKGROUND: Socio-economic disadvantage at both individual and neighbourhood levels has been found to be associated with single lifestyle risk factors. However, it is unknown to what extent their combined effects contribute to a broad lifestyle profile. We aimed to (i) investigate the associations of individual socio-economic disadvantage (ISED) and neighbourhood socio-economic disadvantage (NSED) in relation to an extended score of health-related lifestyle risk factors (lifestyle risk index); and to (ii) investigate whether NSED modified the association between ISED and the lifestyle risk index. METHODS: Of 77 244 participants [median age (IQR): 46 (40-53) years] from the Lifelines cohort study in the northern Netherlands, we calculated a lifestyle risk index by scoring the lifestyle risk factors including smoking status, alcohol consumption, diet quality, physical activity, TV-watching time and sleep time. A higher lifestyle risk index was indicative of an unhealthier lifestyle. Composite scores of ISED and NSED based on a variety of socio-economic indicators were calculated separately. Linear mixed-effect models were used to examine the association of ISED and NSED with the lifestyle risk index and to investigate whether NSED modified the association between ISED and the lifestyle risk index by including an interaction term between ISED and NSED. RESULTS: Both ISED and NSED were associated with an unhealthier lifestyle, because ISED and NSED were both positively associated with the lifestyle risk index {highest quartile [Q4] ISED beta-coefficient [95% confidence interval (CI)]: 0.64 [0.62-0.66], P < 0.001; highest quintile [Q5] NSED beta-coefficient [95% CI]: 0.17 [0.14-0.21], P < 0.001} after adjustment for age, sex and body mass index. In addition, a positive interaction was found between NSED and ISED on the lifestyle risk index (beta-coefficient 0.016, 95% CI: 0.011-0.021, Pinteraction < 0.001), which indicated that NSED modified the association between ISED and the lifestyle risk index; i.e. the gradient of the associations across all ISED quartiles (Q4 vs Q1) was steeper among participants residing in the most disadvantaged neighbourhoods compared with those who resided in the less disadvantaged neighbourhoods. CONCLUSIONS: Our findings suggest that public health initiatives addressing lifestyle-related socio-economic health differences should not only target individuals, but also consider neighbourhood factors.

Circulating de novo lipogenesis fatty acids and all-cause mortality in a prospective Dutch population cohort.

BACKGROUND: Circulating fatty acids (FA) from de novo lipogenesis (DNL) are associated with all-cause mortality in individuals with elevated CVD risk. However, compared to FA early in the DNL synthetic pathway, cis-vaccenic acid, one of the FA distal in the DNL synthetic pathway, has rarely been studied in a general population cohort. We hypothesized that circulating cis-vaccenic acid is more strongly related to all-cause mortality than other circulating DNL-related FA. OBJECTIVES: The primary and secondary objectives of this study were to investigate the prospective associations of plasma levels of cis-vaccenic acid and other DNL-related FA with all-cause mortality in a general population, respectively. METHODS: We included 850 participants (mean ± SD age 53 ± 15 years) from the Dutch Lifelines cohort study. Circulating levels of palmitic (C16:0), palmitoleic (C16:1n7), cis-vaccenic (cis-C18:1n7), stearic (C18:0), oleic acid (C18:1n9) in plasma phospholipids (PL) and triglycerides (TG) were measured by gas chromatography. The associations of circulating cis-C18:1n7 and other DNL-related FA with all-cause mortality were assessed using Cox regression analyses. RESULTS: During a median follow-up of 9.3 (IQR: 5.4-10.8) years, 34 (4.0%) participants had died. In plasma PL, a 1-SD increase in cis-C18:1n7 was associated with an increased risk of all-cause mortality in univariate and multivariate models (p<0.02 for all), with a HR [95% CI] of 1.60 [1.13-2.25] after adjustment for age and sex. CONCLUSIONS: Circulating plasma PL cis-C18:1n7 was associated with a higher risk for all-cause mortality. More studies are needed in different cohorts to verify and validate our results.

Interpretation of Folate Results in Hemolytic Plasma Samples: A Practical Approach.

Folate analysis in plasma is affected by hemolysis, which can lead to biased results. However, the degree of hemolysis that is considered acceptable is unclear. We explored the relationship between folate concentration and degree of hemolysis. Heparin plasma samples (N=77, hemolysis index ≤10 µmol/L) were spiked with increasing amounts of corresponding patient-specific hemolysate. Subsequently, the folate concentration and hemolysis index were measured using two Roche Cobas platforms, and their incremental relationship was investigated. The folate concentration ranged from 2.9 to 30.9 nmol/L with a median (interquartile range) of 11.4 (8.6-19.1) nmol/L. The linear relationship between the increments in folate concentration and hemolysis index was approximated by the function y=1.86x+1.56 (R2=0.996), where x represents the laboratory-specific critical difference in folate concentration, which can be calculated from the analytical variation of the employed folate assay(s), and y represents the hemolysis threshold. The hemolysis threshold did not significantly differ between the tertiles of plasma folate concentration (P=0.10). In conclusion, we have provided an evidence-based approach that can be used to reliably interpret folate concentrations in hemolytic samples, independent of the patient's folate status.

Urinary 3-hydroxyisovaleryl carnitine excretion, protein energy malnutrition and risk of all-cause mortality in kidney transplant recipients: Results from the TransplantLines cohort studies.

BACKGROUND: Leucine is an essential amino acid and a potent stimulator of muscle protein synthesis. Since muscle wasting is a major risk factor for mortality in kidney transplant recipients (KTR), dietary leucine intake might be linked to long-term mortality. Urinary 3-hydroxyisovaleryl carnitine (3-HIC) excretion, a functional marker of marginal biotin deficiency, may also serve as a marker for dietary leucine intake. OBJECTIVE: In this study we aimed to investigate the cross-sectional determinants of urinary 3-HIC excretion and to prospectively investigate the association of urinary 3-HIC excretion with all-cause mortality in KTR. DESIGN: Urinary 3-HIC excretion and plasma biotin were measured in a longitudinal cohort of 694 stable KTR. Cross-sectional and prospective analyses were performed using ordinary least squares linear regression analyses and Cox regression analyses, respectively. RESULTS: In KTR (57% male, 53 ± 13 years, estimated glomerular filtration rate 45 ± 19 mL/min/1.73 m2), urinary 3-HIC excretion (0.80 [0.57-1.16] µmol/24 h) was significantly associated with plasma biotin (std. ß = -0.17; P < 0.001). Subsequent adjustment for potential covariates revealed urinary creatinine excretion (std. ß = 0.24; P < 0.001) and urinary urea excretion (std. ß = 0.53; P < 0.001) as the primary determinant of urinary 3-HIC excretion. Whereas plasma biotin explained only 1% of the variance in urinary 3-HIC excretion, urinary urea excretion explained >45%. During median follow-up for 5.4 [4.8-6.1] years, 150 (22%) patients died. Log2-transformed urinary 3-HIC excretion was inversely associated with all-cause mortality (HR: 0.52 [0.43-0.63]; P < 0.001). This association was independent of potential confounders. CONCLUSIONS: Urinary 3-HIC excretion more strongly serves as a marker of leucine intake than of biotin status. A higher urinary 3-HIC excretion is associated with a lower risk of all-cause mortality. Future studies are warranted to explore the underlying mechanism. TRIAL REGISTRATION ID: NCT02811835. TRIAL REGISTRATION URL: https://clinicaltrials.gov/ct2/show/NCT02811835.

Urinary Excretion of 6-Sulfatoxymelatonin, the Main Metabolite of Melatonin, and Mortality in Stable Outpatient Renal Transplant Recipients.

Melatonin is a multifaceted hormone which rises upon the onset of darkness. Pineal synthesis of melatonin is known to be disturbed in patients with end-stage renal disease, but it is not known if its production is restored to normal after successful renal transplantation. We hypothesized that urinary excretion of 6-sulfatoxymelatonin, the major metabolite of melatonin, is lower in renal transplant recipients (RTRs) compared to healthy controls and that this is associated with excess mortality. Urinary 6-sulfatoxymelatonin was measured via LC-MS/MS in 701 stable outpatient RTRs and 285 healthy controls. Median urinary 6-sulfatoxymelatonin in RTR was 13.2 nmol/24h, which was 47% lower than in healthy controls. Urinary 6-sufatoxymelatonin appeared undetectable in the majority of 36 RTRs with diabetic nephropathy as primary renal disease. Therefore, this subgroup was excluded from further analyses. Of the remaining 665 RTRs, during 5.4 years of follow-up, 110 RTRs died, of whom 38 died due to a cardiovascular cause. In Cox-regression analyses, urinary 6-sulfatoxymelatonin was significantly associated with all-cause mortality (0.60 (0.44-0.81), p = 0.001) and cardiovascular mortality (0.49 (0.29-0.84), p = 0.009), independent of conventional risk factors and kidney function parameters. Based on these results, evaluation and management of melatonin metabolism could be considered for improvement of long-term outcomes in RTRs.