Associations of perchlorate, nitrate, and thiocyanate exposure with arthritis and inflammation indicators in young and middle-aged adults, NHANES 2005-2016.

Background: Perchlorates, nitrates, and thiocyanates are prevalent environmental chemicals. Their potential association with arthritis remains unexplored. This study aimed to investigate the link between perchlorate, nitrate, and thiocyanate exposure and arthritis, as well as the potential role of inflammation in this context. Methods: Utilizing the National Health and Nutrition Examination Survey (NHANES) data spanning from 2005 to 2016, the study enrolled 6597 participants aged 20-59 (young and middle-aged), of which 1045 had arthritis. Employing multivariate logistic regression modeling, multiple linear regression models, restricted cubic spline analysis, Bayesian kernel machine regression (BKMR) modeling, and mediation analysis, we assessed these relationships. Results: There was a significant positive association between elevated urinary thiocyanate levels and arthritis risk [1.19 (1.11, 1.28)]. This association held true across subgroups of osteoarthritis (OA) [1.24 (1.10, 1.40)] and rheumatoid arthritis (RA) [1.33 (1.15, 1.55)]. Thiocyanate levels displayed a dose-dependent relationship with arthritis risk, showing a linear trend (nonlinear P > 0.05). Conversely, perchlorate and nitrate did not exhibit associations with arthritis risk. BKMR outcomes highlighted a positive correlation between a mixture of perchlorate, nitrate, and thiocyanate and arthritis risk, with thiocyanate being the predominant predictors. Moreover, BKMR and generalized linear model analyses unveiled no significant synergistic effect of urinary perchlorate, nitrate, and thiocyanate on arthritis risk. Furthermore, thiocyanate exposure has been linked to elevated levels of inflammatory indicators (white blood cell, neutrophils, lymphocytes, and systemic immune-inflammatory index (SII)). Conclusion: Heightened thiocyanate exposure may be linked to elevated arthritis risk, either single or in combined effects. Additionally, thiocyanate exposure is associated with heightened inflammation levels.

Associations of Trimester-Specific Exposure to Perchlorate, Thiocyanate, and Nitrate with Childhood Neurodevelopment: A Birth Cohort Study in China.

Studies about the impacts of maternal exposure to perchlorate, thiocyanate, and nitrate on offspring neurodevelopment are scarce. Based on a birth cohort in China, 1,028 mothers provided urine samples at three trimesters for determination of the three target analytes, and their offspring neurodevelopment was evaluated at 2 years old. Associations of maternal exposure to the three chemicals with offspring neurodevelopment were estimated using three statistical methods. Trimester-specific analyses using generalized estimating equation models showed that double increment of thiocyanate and nitrate during the first trimester was associated with 1.56 (95% CI: -2.82, -0.30) and 1.22 (-2.40, -0.03) point decreases in the offspring mental development index (MDI), respectively. Weighted quantile sum (WQS) regression analyses showed that the mixture exposure at the first and second trimesters was negatively associated with the offspring MDI (ß = -2.39, 95% CI: -3.85, -0.93; ß = -1.75, 95% CI: -3.04, -0.47, respectively) and thiocyanate contributed the most to the association (65.0 and 91.6%, respectively). Bayesian kernel machine regression analyses suggested an inverted U-shape relationship of maternal urinary thiocyanate with the offspring MDI. These findings suggested that prenatal exposure to the three chemicals (at current levels), especially thiocyanate and nitrate, may impair neurodevelopment. Early pregnancy seems to be the sensitive window.

Urinary perchlorate, thiocyanate, and nitrate and their associated risk factors among Chinese pregnant women.

Perchlorate, nitrate, and thiocyanate are well-known inhibitors of iodide uptake and thyroid-disrupting chemicals. Widespread human exposure to them has been identified, whereas studies on their internal exposure levels among Chinese pregnant women are scarce and factors associated with them are not well recognized. The objective of this study is to determine their levels and identify the associated factors among pregnant women (n = 1120), based on a prospective birth cohort in Wuhan, central China, using repeated urine samples of three trimesters. Urinary perchlorate, thiocyanate, and nitrate were 100% detected in the samples, and specific gravity-adjusted median concentrations of them in all the samples were 12.6 ng/mL, 367 ng/mL, and 63.7 µg/mL, respectively. Their concentrations were weakly-to-moderately correlated with each other, with Spearman correlation coefficients ranging from 0.27 to 0.54. Poor reproducibility were observed for the three analytes over the three trimesters, with intraclass correlation coefficient of 0.07, 0.19, 0.04 for perchlorate, thiocyanate, and nitrate, respectively. The women who were overweight or used tap water as drinking water had significantly higher perchlorate concentrations, while those with excessive gestational weight gain had significantly higher thiocyanate concentrations (p < 0.05). The women with a college degree or above had lower nitrate concentrations (p < 0.05). Meanwhile, the median concentration of perchlorate in urine samples collected in spring, thiocyanate in those collected in winter, and nitrate in those collected in autumn, was significantly higher compared to their median concentrations in the samples collected in other three seasons (p < 0.05), respectively. Urinary perchlorate and nitrate concentrations of pregnant women in this study were higher than the concentrations of pregnant women in other countries, while thiocyanate concentrations were lower than that of most other countries. This study suggested potential covariates for future epidemiological analyses.

Effects of perchlorate, nitrate, and thiocyanate exposures on serum total testosterone in children and adolescents.

Perchlorate, nitrate, and thiocyanate are common thyroid disruptors in daily life and alter testosterone levels in animals. However, little is known about the effects of perchlorate, nitrate, and thiocyanate on serum total testosterone (TT) in the general population. The study was designed to assess the associations between urinary levels of perchlorate, nitrate, and thiocyanate and serum total testosterone (TT) in the general population. The present study utilized data from the 2011-2016 National Health and Nutritional Examination Survey (NHANES). A total of 6201 participants aged 6-79 with information on urinary perchlorate, nitrate, thiocyanate, and serum total testosterone were included. We conducted multiple linear regression models and Bayesian Kernel Machine Regression (BKMR) models to estimate the associations by sex-age groups. Children (ages 6-11) have higher levels of perchlorate and nitrate than the rest. After adjusting for covariates, urinary perchlorate was significantly negatively associated with serum TT in male adolescents (ß = -0.1, 95 % confidence interval: -0.2, -0.01) and female children [-0.13, (-0.21, -0.05)]. Urinary nitrate was significantly negatively associated with serum TT in female children, while urinary thiocyanate was significantly positively associated with serum TT in female adults aged 20 to 49 [0.05 (0.02, 0.08)]. BKMR analysis indicated that no other interactions were found between urinary perchlorate, nitrate, and thiocyanate. Our findings suggested that urinary perchlorate, nitrate, and thiocyanate levels may relate to serum total testosterone levels in specific sex-age groups. We identified male adolescents and female children as are most sensitive subgroups where testosterone is susceptible to interference.

Association between environmental exposure to perchlorate, nitrate, and thiocyanate and serum α-Klotho levels among adults from the National Health and nutrition examination survey (2007-2014).

BACKGROUND & AIMS: Aging is a pathophysiological process driven by a diverse set of complex biological processes, and environmental pollution plays an important role in this process. This study aimed to explore the association between serum α-Klotho levels and urinary perchlorate, nitrate, and thiocyanate levels. METHODS: This secondary dataset analysis included 4875 participants (mean age, 57.69 year; male, 49.58%; non-Hispanic White, 47.67%) from the US National Health and Nutrition Examination Survey (2007-2014). Enzyme-linked immunosorbent assay was used to quantify α-Klotho levels, and ion chromatography coupled with electrospray tandem mass spectrometry was used to quantify thiocyanate, nitrate, and perchlorate levels. Multivariate linear regression models were applied to estimate the association between perchlorate, nitrate, and thiocyanate levels and serum α-Klotho levels. RESULTS: Urinary thiocyanate levels were negatively associated with α-Klotho levels (ß = - 0.006; 95% confidence interval, - 0.010 to - 0.003; P = 0.0004) after adjusting for age, sex, body mass index, race, alcohol consumption, estimated glomerular filtration rate, underlying disease, physical activity, smoking status, usual energy intake, and urinary creatinine and serum cotinine levels and mutual adjustment of urinary perchlorate, urinary nitrate, and urinary thiocyanate levels. The α-Klotho level in participants in the highest quartile was higher by 50.567 ng/mL (ß = 50.567; 95% confidence interval, 14.407 to 86.726; P = 0.009) than that in participants in the lowest quartile of urinary perchlorate. A linear relationship was observed between urinary thiocyanate and α-Klotho levels. CONCLUSIONS: Urinary thiocyanate levels were negatively associated with serum α-Klotho levels. Urinary thiocyanate should be further investigated as a potential mediator of aging and age-related diseases.

The Association Between Environmental Perchlorate, Nitrate, and Thiocyanate Exposure and Oral Pain in NHANES.

Aim: To examine the human exposure to perchlorate, nitrate, and thiocyanate, and their associations with oral pain (OP) in the general population from the U.S. Methods: A total of 13,554 participants were enrolled in the National Health and Nutrition Examination Survey. The urinary perchlorate, nitrate, and thiocyanate were measured using ion chromatography coupled with an electrospray tandem mass spectrometry. The multivariable linear and logistic regressions were performed to explore the associations of the urinary perchlorate, nitrate, and thiocyanate, with the prevalence of oral pain. Restricted cubic splines were used to explore the non-linearity. Results: There are 3,129 OP cases. There was a higher urinary level of perchlorate, nitrate, and thiocyanate in OP. We found that urinary thiocyanate was positively associated with OP (odds ratio [OR] = 1.06; [1, 1.13]; p = 0.049). Restricted cubic spines revealed that urinary thiocyanate was in a U-shape association with OP. Conclusions: Urinary thiocyanate was in a U-shape association with OP, suggesting that we should keep the exposure of thiocyanate under a reasonable range.

Design and Functionalization of a µPAD for the Enzymatic Determination of Nitrate in Urine.

In this work, the design of a microfluidic paper-based analytical device (µPAD) for the quantification of nitrate in urine samples was described. Nitrate monitoring is highly relevant due to its association to some diseases and health conditions. The nitrate determination was achieved by combining the selectivity of the nitrate reductase enzymatic reaction with the colorimetric detection of nitrite by the well-known Griess reagent. For the optimization of the nitrate determination µPAD, several variables associated with the design and construction of the device were studied. Furthermore, the interference of the urine matrix was evaluated, and stability studies were performed, under different conditions. The developed µPAD enabled us to obtain a limit of detection of 0.04 mM, a limit of quantification of 0.14 mM and a dynamic concentration range of 0.14-1.0 mM. The designed µPAD proved to be stable for 24 h when stored at room temperature in air or vacuum atmosphere, and 60 days when stored in vacuum at -20 °C. The accuracy of the nitrate µPAD measurements was confirmed by analyzing four certified samples (prepared in synthetic urine) and performing recovery studies using urine samples.

Sex-Dependent Effects of Nephron Ift88 Disruption on BP, Renal Function, and Cystogenesis.

BACKGROUND: Primary cilia regulation of renal function and BP in health and disease is incompletely understood. This study investigated the effect of nephron ciliary loss on renal physiology, BP, and ensuing cystogenesis. METHODS: Mice underwent doxycycline (DOX)-inducible nephron-specific knockout (KO) of the Ift88 gene at 2 months of age using a Cre-LoxP strategy. BP, kidney function, and renal pathology were studied 2 and 9 months after DOX (Ift88 KO) or vehicle (control). RESULTS: At 2 months post-DOX, male, but not female, Ift88 KO, compared with sex-matched control, mice had reduced BP, enhanced salt-induced natriuresis, increased urinary nitrite and nitrate (NOx) excretion, and increased kidney NOS3 levels, which localized to the outer medulla; the reductions in BP in male mice were prevented by L-NAME. At 9 months post-DOX, male, but not female, Ift88 KO mice had polycystic kidneys, elevated BP, and reduced urinary NOx excretion. No differences were observed in plasma renin concentration, plasma aldosterone, urine vasopressin, or urine PGE2 between Ift88 KO and control mice at 2 or 9 months post-DOX. CONCLUSIONS: Nephron cilia disruption in male, but not female, mice (1) reduces BP prior to cyst formation, (2) increases NOx production that may account for the lower BP prior to cyst formation, and (3) induces polycystic kidneys that are associated with hypertension and reduced renal NO production.

Relationship between urinary nitrate concentrations and cognitive function in older adults: findings from the NHANES survey.

This study evaluated the association of urinary nitrate concentrations with cognition in older subjects enrolled in the NHANES study. We also explored whether associations between urinary nitrate and cognition were modified by cardiovascular risk, vitamin D status and vitamin C intake. Two NHANES cycles were merged (2011-2012 and 2013-2014) and a total of 1,015 adults aged 60-80 (69.4 ± 0.3) years were included. Cognition was assessed using the Word List Learning, Word List Recall, Animal Fluency and the Digit Symbol Substitution tests. Urinary nitrate was analysed using electrospray tandem mass spectrometry. Urinary nitrate concentrations were not associated with cognitive performance on any of the cognitive tests. Associations were also not significant in subjects at greater risk for cognitive impairment (i.e. high cardiovascular risk and non-optimal vitamin D status). Longitudinal analyses are needed to explore the associations of urinary nitrate concentrations with dietary nitrate intake and cognitive function.

Physical Training Is a Potential Modifier of Risk for Contrast-Induced Acute Kidney Injury in Diabetes Mellitus.

BACKGROUND: Iodinated contrast (IC) is a leading cause of hospital-based acute kidney injury (AKI). Contrast-induced acute kidney injury (CI-AKI) is a decline in renal function due to iodinated contrast administration and occurs more frequently in individuals with increasingly common risk factors, such as diabetes mellitus (DM). Physical training (PT) can have renoprotective effects on CI-AKI in diabetic nephropathy. The aim of this study was to evaluate the injury in kidneys of diabetic rats submitted to treatment with IC, evaluating the impact of PT on hemodynamics and renal function in addition to oxidative profile in diabetic rats submitted to IC-AKI. MATERIALS AND METHODS: Adult male Wistar rats are randomized into four groups: citrate (n = 7): control group, citrate buffer (streptozotocin-STZ vehicle), intravenous tail (iv), single dose; DM (n = 7): STZ, 60 mg/kg, iv, single dose; DM+IC (n = 7): DM rats treated with IC (sodium meglumine ioxithalamate, 6 mL/kg, intraperitoneal (ip), single dose); DM+IC+PT (n = 7): DM rats treated with IC as mentioned and submitted to physical training. Renal function parameters (inulin clearance, neutrophil gelatinase-associated lipocalin (NGAL), serum creatinine, and urinary albumin), hemodynamics (renal blood flow and renal vascular resistance), and oxidative profile (urinary peroxides, urinary TBARS, urinary nitric oxide, and renal tissue thiols) were evaluated. RESULTS: It was possible to observe a decrease in inulin clearance, renal blood flow, and thiols in renal tissue accompanied by an increase in urinary flow, serum creatinine, urinary albumin, renal vascular resistance, urinary peroxides, urinary nitrate, and TBARS in the DM group compared to the citrate group. The DM+IC group showed a reduction in inulin clearance, and the renal dysfunction was also seen by the increased NGAL. Renal hemodynamics and oxidative profile compared were also worsened in the DM group. PT improved renal function by increasing renal blood flow and thiol levels in renal tissue and reduced renal vascular resistance, metabolites of reactive oxygen, nitrogen species, and lipid peroxidation in the DM+IC+PT group compared to DM+IC. CONCLUSIONS: Our results confirmed that DM induction increases renal vulnerability to the toxicity of IC and an association between DM with IC predisposes to severe AKI with reduced renal function alongside with renal hemodynamic alterations and oxidative mechanism of injury. The PT showed a renoprotective effect in DM animals subjected to damage with IC by modulating renal hemodynamics and oxidative profile, confirming a potential to modify the risk of CI-AKI when diabetes mellitus is present.

Higher urinary nitrate was associated with lower prevalence of congestive heart failure: results from NHANES.

BACKGROUND: Some studies have reported that nitrate intake from vegetables was inversely associated with many vascular diseases, but few studies have paid attention to the relationship between urinary nitrate and cardiovascular diseases (CVDs). This cross-sectional study aimed to explore the connections between urinary nitrate and prevalence of CVDs. METHODS: The data of this study was collected from National Health and Nutrition Examination Survey (NHANES). Finally, several years' data of NHANES were merged into 14,894 observations. Logistic regression models were used to examine the associations between urinary nitrate and CVDs by using the "survey" package in R software (version 3.2.3). RESULTS: In the univariable logistic analysis, significant association was discovered between urinary nitrate and congestive heart failure, coronary heart disease, angina pectoris, myocardial infarction (all P < 0.001). By adjusting related covariates, the multivariable logistic analysis showed that the significant association only existed between urinary nitrate and congestive heart failure (OR = 0.651, 95% CI 0.507-0.838, P < 0.001). Compared to Q1 urinary nitrate level as reference, the risk for prevalent heart failure diminished along with increasing levels of urinary nitrates, (OR of Q2 level = 0.633, 95% CI 0.403-0.994), (OR of Q3 level = 0.425, 95% CI 0.230-0.783), (OR of Q4 level = 0.375, 95% CI 0.210-0.661), respectively. Moreover, urinary nitrate levels were associated with congestive heart failure in a dose-dependent manner in both 20-60 years group, 60+ years group and male, female group (P < 0.001, P = 0.011 and P = 0.009, P = 0.004). CONCLUSIONS: Independent of related covariates, higher urinary nitrate was associated with lower prevalent congestive heart failure.

Urinary nitrate concentration as a marker for kidney transplant rejection.

BACKGROUND: Early identification and treatment of kidney transplant rejection episodes is vital to limit loss of function and prolong the life of the transplanted kidney and recipient. Current practice depends on detecting a creatinine rise. A biomarker to diagnose transplant rejection at an earlier time point than current practice, or to inform earlier decision making to biopsy, could be transformative. It has previously been shown that urinary nitrate concentration is elevated in renal transplant rejection. Nitrate is a nitric oxide (NO) oxidation product. Transplant rejection upregulates NO synthesis via inducible nitric oxide synthase leading to elevations in urinary nitrate concentration. We have recently validated a urinary nitrate concentration assay which could provide results in a clinically relevant timeframe. Our aim was to determine whether urinary nitrate concentration is a useful tool to predict renal transplant rejection in the context of contemporary clinical practice. METHODS: We conducted a prospective observational study, recruiting renal transplant participants over an 18-month period. We made no alterations to the patients' clinical care including medications, immunosuppression, diet and frequency of visits. We collected urine samples from every clinical attendance. We assessed the urinary nitrate to creatinine ratio (uNCR) between patient groups: routine attendances, biopsy proven rejection, biopsy proven no rejection and other call backs. uNCR was examined over time for those with biopsy proven transplant rejection. These four groups were compared using an ANOVA test. RESULTS: A total of 2656 samples were collected. uNCR during biopsy proven rejection, n = 15 (median 49 µmol/mmol, IQR 23-61) was not significantly different from that of routine samples, n = 164 (median 55 µmol/mmol, IQR 37-82) (p = 0.55), or biopsy proven no rejection, n = 12 (median 39 µmol/mmol, IQR 21-89) (P = 0.77). Overall uNCR was highly variable with no diagnostic threshold for kidney transplant rejection. Furthermore, within-patient uNCR was highly variable over time, and thus it was not possible to produce individualised patient thresholds to identify rejection. The total taking Tacrolimus was 204 patients, with no statistical difference between the uNCR of all those on Tacrolimus, against those not, p = 0.18. CONCLUSION: The urinary nitrate to creatinine ratio is not a useful biomarker for renal transplant rejection.

Environment-Wide Association Study of CKD.

BACKGROUND AND OBJECTIVES: Exposure to environmental chemicals has been recognized as one of the possible contributors to CKD. We aimed to identify environmental chemicals that are associated with CKD. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We analyzed the data obtained from a total of 46,748 adults who participated in the National Health and Nutrition Examination Survey (1999-2016). Associations of chemicals measured in urine or blood (n=262) with albuminuria (urine albumin-to-creatinine ratio ≥30 mg/g), reduced eGFR (<60 ml/min per 1.73 m2), and a composite of albuminuria or reduced eGFR were tested and validated using the environment-wide association study approach. RESULTS: Among 262 environmental chemicals, seven (3%) chemicals showed significant associations with increased risk of albuminuria, reduced eGFR, or the composite outcome. These chemicals included metals and other chemicals that have not previously been associated with CKD. Serum and urine cotinines, blood 2,5-dimethylfuran (a volatile organic compound), and blood cadmium were associated with albuminuria. Blood lead and cadmium were associated with reduced eGFR. Blood cadmium and lead and three volatile compounds (blood 2,5-dimethylfuran, blood furan, and urinary phenylglyoxylic acid) were associated with the composite outcome. A total of 23 chemicals, including serum perfluorooctanoic acid, seven urinary metals, three urinary arsenics, urinary nitrate and thiocyanate, three urinary polycyclic aromatic hydrocarbons, and seven volatile organic compounds, were associated with lower risks of one or more manifestations of CKD. CONCLUSIONS: A number of chemicals were identified as potential risk factors for CKD among the general population.

Renomedullary Interstitial Cell Endothelin A Receptors Regulate BP and Renal Function.

BACKGROUND: The physiologic role of renomedullary interstitial cells, which are uniquely and abundantly found in the renal inner medulla, is largely unknown. Endothelin A receptors regulate multiple aspects of renomedullary interstitial cell function in vitro. METHODS: To assess the effect of targeting renomedullary interstitial cell endothelin A receptors in vivo, we generated a mouse knockout model with inducible disruption of renomedullary interstitial cell endothelin A receptors at 3 months of age. RESULTS: BP and renal function were similar between endothelin A receptor knockout and control mice during normal and reduced sodium or water intake. In contrast, on a high-salt diet, compared with control mice, the knockout mice had reduced BP; increased urinary sodium, potassium, water, and endothelin-1 excretion; increased urinary nitrite/nitrate excretion associated with increased noncollecting duct nitric oxide synthase-1 expression; increased PGE2 excretion associated with increased collecting duct cyclooxygenase-1 expression; and reduced inner medullary epithelial sodium channel expression. Water-loaded endothelin A receptor knockout mice, compared with control mice, had markedly enhanced urine volume and reduced urine osmolality associated with increased urinary endothelin-1 and PGE2 excretion, increased cyclooxygenase-2 protein expression, and decreased inner medullary aquaporin-2 protein content. No evidence of endothelin-1-induced renomedullary interstitial cell contraction was observed. CONCLUSIONS: Disruption of renomedullary interstitial cell endothelin A receptors reduces BP and increases salt and water excretion associated with enhanced production of intrinsic renal natriuretic and diuretic factors. These studies indicate that renomedullary interstitial cells can modulate BP and renal function under physiologic conditions.

Nitric oxide-related markers link inversely to blood pressure in black boys and men: the ASOS and African-PREDICT studies.

Nitric oxide plays an important role in maintaining endothelial function, while increased oxidative stress may lead to nitric oxide inactivation and cardiovascular disease. If nitric oxide biosynthesis/bioavailability is already suppressed early in life, it may potentially predispose an individual to the early development of cardiovascular disease. We therefore aimed to identify differences in nitric oxide-related markers (urinary nitrate, nitrite and the nitrate-to-nitrite ratio (UNOxR)) between young black and white individuals, and whether these markers are associated with blood pressure and carotid intima media thickness. We included black and white healthy boys (n = 80; aged 6-8 years) and men (n = 510; 20-30 years) and measured blood pressure and carotid intima media thickness, along with urinary biochemical markers including nitrate and nitrite. The black boys and men had lower nitrate and UNOxR (all p ≤ 0.003) than their white counterparts. In single and multiple regression analyses, we found an inverse association of diastolic blood pressure in the black boys (adj. R2 = 0.27; ß = -0.32; p = 0.030), and systolic blood pressure in black men (adj. R2 = 0.07; ß = -0.13; p = 0.036) with nitrate. Carotid intima media thickness associated inversely with UNOxR in the black men (adj. R2 = 0.02; ß = -0.14; p = 0.023), but not in the boys. Lower urinary nitrate in black boys and young men was associated negatively with blood pressure, suggesting that potentially lower nitric oxide bioavailability in young black individuals may contribute to hypertension development in later life.

Renal nitrate clearance in chronic kidney disease.

BACKGROUND: Nitric oxide (NO) is rapidly oxidised in humans to nitrite and nitrate, with nitrate being present in much greater abundance. These oxidation products can be recycled back into nitric oxide via a complex entero-salivary pathway, thus preserving NO activity. Approximately 65% of circulating nitrate is excreted in the urine in 48 h, with the excretory pathway of the remainder unknown. The effect of declining renal function on nitrate clearance is unknown METHODS: Forty five subjects, 21 M, 24F, median age 69 (range 27-75 years) with renal function assessed by CKD-EPI eGFR between 9 and 89 ml/min/1.73 m2 completed the study. Following a 24 h low nitrate diet a microplate spectrophotometric method was employed to measure plasma nitrate concentration and 24 h urinary nitrate excretion were measured to determine renal nitrate clearance. RESULTS: There was a strong positive correlation between urinary nitrate clearance and eGFR, (Spearman R = 0.7665, p < 0.0001) with a moderate negative correlation between plasma nitrate concentration and CKD-EPI eGFR, (Spearman's R = -0.37, p = 0.012). There was a trend between fractional excretion of nitrate and CKD-EPI eGFR (ml/min/1.73 m2) Spearman's R 0.27, p = 0.07 though this did not reach statistical significance. Plasma nitrate concentration and serum creatinine concentration were positively correlated, Spearman's R = 0.39, p = 0.008. CONCLUSIONS: We have observed a strong positive association between renal nitrate clearance and renal function such that plasma nitrate rises as renal function falls. Fractional excretion of nitrate appears to decline as renal function falls. As such, urinary nitrate excretion is unlikely to be a reliable marker of endogenous NO synthesis in settings where renal function is altered.

Maternal undernutrition aggravates renal tubular necrosis and interstitial fibrosis after unilateral ureteral obstruction in male rat offspring.

Maternal undernutrition is known to reduce glomerular number but it may also affect tubulointerstitium, capillary density, and response to oxidative stress. To investigate whether the latter elements are affected, we examined the response to unilateral ureteral obstruction (UUO), an established model of renal tubulointerstitial fibrosis, in the kidney of offspring from control and nutrient restricted rats. Six-week old male offspring from rats given food ad libitum (CON) and those subjected to 50% food restriction throughout pregnancy (NR) were subjected to UUO for 7 days. Body weight was significantly lower in NR. Systolic blood pressure and blood urea nitrogen increased similarly in CON and NR after UUO. Tubular necrosis in the obstructed kidney, on the other hand, was more extensive in NR. Also, the collagen area, a marker of fibrosis, of the obstructed kidney was significantly increased compared with the contralateral kidney only in NR. Capillary density was decreased similarly in the obstructed kidney of CON and NR compared with the contralateral kidney. Urine nitrate/nitrite, a marker of nitric oxide production, from the obstructed kidney was significantly increased in NR compared with CON. Nitrotyrosine, a marker of nitric oxide-mediated free radical injury, was increased in the obstructed kidney compared with the contralateral kidney in both CON and NR, but the extent was significantly greater in NR. In conclusion, more severe tubular necrosis and fibrosis after UUO was observed in NR, which was thought to be due to increased nitrosative stress.

Consumption of Nitrate-Rich Beetroot Juice with or without Vitamin C Supplementation Increases the Excretion of Urinary Nitrate, Nitrite, and N-nitroso Compounds in Humans.

Consumption of nitrate-rich beetroot juice (BRJ) by athletes induces a number of beneficial physiological health effects, which are linked to the formation of nitric oxide (NO) from nitrate. However, following a secondary pathway, NO may also lead to the formation of N-nitroso compounds (NOCs), which are known to be carcinogenic in 39 animal species. The extent of the formation of NOCs is modulated by various other dietary factors, such as vitamin C. The present study investigates the endogenous formation of NOCs after BRJ intake and the impact of vitamin C on urinary NOC excretion. In a randomized, controlled trial, 29 healthy recreationally active volunteers ingested BRJ with or without additional vitamin C supplements for one week. A significant increase of urinary apparent total N-nitroso Compounds (ATNC) was found after one dose (5 to 47 nmol/mmol: p < 0.0001) and a further increase was found after seven consecutive doses of BRJ (104 nmol/mmol: p < 0.0001). Vitamin C supplementation inhibited ATNC increase after one dose (16 compared to 72 nmol/mmol, p < 0.01), but not after seven daily doses. This is the first study that shows that BRJ supplementation leads to an increase in formation of potentially carcinogenic NOCs. In order to protect athlete's health, it is therefore important to be cautious with chronic use of BRJ to enhance sports performances.

GC-MS measurement of biological NG-hydroxy-L-arginine, a stepmotherly investigated endogenous nitric oxide synthase substrate and arginase inhibitor.

L-Arginine is converted by nitric oxide synthase (NOS) to L-citrulline and nitric oxide (NO). NG-Hydroxy-L-arginine (NOHA) is the isolable intermediate of this reaction. NOHA has been identified in biological samples by gas chromatography-mass spectrometry (GC-MS) and quantified by high-performance liquid chromatography (HPLC). Reportedly, NOHA concentrations in human plasma and serum range over four orders of magnitude (e.g., 2 nM-34 µM). The natural occurrence of NOHA in urine has not been reported thus far. Here, we report a validated stable-isotope dilution GC-MS method for the quantitative determination of NOHA in 10-µL aliquots of human serum and urine samples. The method is based on a two-step derivatization of NOHA to the methyl ester pentafluoropropionyl (PFP) derivatives using newly synthesized trideuteromethyl ester NOHA (d3Me-NOHA) as the internal standard and GC-MS quantification. NOHA was found to form a methyl ester-NG,Nδ,Nα-pentafluoropropionyl derivative, i.e., Me-(PFP)3 (M, 642) with the NG-hydroxy group remaining non-derivatized. Selected-ion monitoring of mass-to-charge (m/z) ratio of 458 for endogenous NOHA and m/z 461 for d3Me-NOHA in the negative-ion chemical ionization mode revealed NOHA concentrations of the order of 0.2 µM in human serum and 3 µM in urine samples. Accuracy (recovery, %) was 91.6 ± 1.6% in serum and 39.9 ± 4.5% in urine. Inorganic nitrate was found to decrease NOHA recovery from urine presumably through the reaction of the OH group of NOHA with nitric acid. Imprecision (RSD,  %) ranged between 1.4 and 14.8% in serum, and between 5.3 and 18.4% in urine in the investigated concentration range (0-15 µM NOHA). Ten healthy kidney donors excreted in the urine (mean ± SEM) 13.9 ± 1.81 µmol NOHA per day before and 10.9 ± 1.4 µmol NOHA per day after kidney donation (P = 0.24). Similar results were observed for dimethylamine (DMA), the major urinary metabolite of asymmetric dimethylarginine (ADMA). Changes in NOHA and DMA correlated positively (r = 0.718, P = 0.019). This is the first report on the occurrence and measurement of NOHA in human urine and on the effect of human unilateral nephrectomy on urinary NOHA and DMA. Healthy kidney donation may be useful as a model for kidney disease.

An engineered E. coli Nissle improves hyperammonemia and survival in mice and shows dose-dependent exposure in healthy humans.

The intestine is a major source of systemic ammonia (NH3); thus, capturing part of gut NH3 may mitigate disease symptoms in conditions of hyperammonemia such as urea cycle disorders and hepatic encephalopathy. As an approach to the lowering of blood ammonia arising from the intestine, we engineered the orally delivered probiotic Escherichia coli Nissle 1917 to create strain SYNB1020 that converts NH3 to l-arginine (l-arg). We up-regulated arginine biosynthesis in SYNB1020 by deleting a negative regulator of l-arg biosynthesis and inserting a feedback-resistant l-arg biosynthetic enzyme. SYNB1020 produced l-arg and consumed NH3 in an in vitro system. SYNB1020 reduced systemic hyperammonemia, improved survival in ornithine transcarbamylase-deficient spfash mice, and decreased hyperammonemia in the thioacetamide-induced liver injury mouse model. A phase 1 clinical study was conducted including 52 male and female healthy adult volunteers. SYNB1020 was well tolerated at daily doses of up to 1.5 × 1012 colony-forming units administered for up to 14 days. A statistically significant dose-dependent increase in urinary nitrate, plasma 15N-nitrate (highest dose versus placebo, P = 0.0015), and urinary 15N-nitrate was demonstrated, indicating in vivo SYNB1020 activity. SYNB1020 concentrations reached steady state by the second day of dosing, and excreted cells were alive and metabolically active as evidenced by fecal arginine production in response to added ammonium chloride. SYNB1020 was no longer detectable in feces 2 weeks after the last dose. These results support further clinical development of SYNB1020 for hyperammonemia disorders including urea cycle disorders and hepatic encephalopathy.