Simultaneous determination of phosphatidylcholine-derived quaternary ammonium compounds by a LC-MS/MS method in human blood plasma, serum and urine samples.

The determination of circulating trimethylamine-N-oxide (TMAO), choline, betaine, l-carnitine and O-acetyl-l-carnitine concentration in different human matrices is of great clinical interest. Recent results highlighted the prognostic value of TMAO and quaternary ammonium containing metabolites in the field of cardiovascular and kidney diseases. Herein, we report a method for the rapid and simultaneous measurement of closely related phosphatidylcholine-derived metabolites in three different biological matrices by stable isotope dilution assay. Plasma, serum and urine samples were simply deproteinized and separated by HILIC-chromatography. Detection and quantification were performed using LC-MS/MS with electrospray ionization in positive mode. For accuracy and precision, full calibration was performed covering more than the full reference range. Assay performance metrics include intra- and interday imprecision were below 10% for all analytes. To exclude matrix effects standard addition methods were applied for all matrices. It was shown that calibration standards and quality control prepared in water can be used instead of matrix-matched calibration and controls. The LC/MS/MS-based assay described in this article may improve future clinical studies evaluating TMAO and related substances as prognostic markers for cardiovascular risk and all-cause mortality in different patient populations.

Perchlorate exposure and thyroid function in ammonium perchlorate workers in Yicheng, China.

The impact of low level dust on the thyroid function of workers chronically exposed to ammonium perchlorate (AP) is uncertain and controversial. The aim of this study was to examine whether workers in China with long-term (>3 years) occupational exposure to low levels of AP dust had affected thyroid homeostasis. Mean occupational exposures to AP dust ranged from 0.43 to 1.17 mg/m3. Geometric means of post-shift urinary perchlorate levels were 20.5 µg/L for those exposed and 12.8 µg/L for the controls. No significant differences were found for thyroid function parameters of FT3, FT4, or log TSH or for TPO prevalence or thyroglobulin levels. Additionally, no differences in findings were observed for complete blood count (CBC), serum biochemical profile, or pulmonary function test. Median urinary iodine levels of 172 and 184 µg/L showed that the workers had sufficient iodine intake. This study found no effect on thyroid function from long term, low-level documented exposure to ammonium perchlorate. It is the first study to report both thyroid status parameters and urinary perchlorate, a biomarker of internal perchlorate exposure, in occupationally exposed workers in China.

Direct urine ammonium measurement: time to discard urine anion and osmolar gaps.

BACKGROUND: A failure of urine ammonium to increase during acidosis indicates impaired renal acidification, and the urinary ammonium concentration is therefore a useful investigation in determining the cause of a metabolic acidosis. However, urine ammonium measurements are not widely available in routine diagnostic laboratories. This has led to the use of urine anion or osmolar gaps, which are unsatisfactory as surrogates for urine ammonium measurement. METHODS: We evaluated the adaptation of two widely available automated plasma ammonium assays for measurement of urinary ammonium. RESULTS: Both assays showed good recovery and linearity in urine samples spiked with ammonium chloride, and acceptable precision. Urine ammonium concentrations estimated from urinary anion and osmolar gaps showed poor agreement with measured urine ammonium concentrations. CONCLUSIONS: Direct urine ammonium measurements are easily performed with modern autoanalysers by simple adaptation of routine plasma ammonium assays. The use of urine anion and osmolar gaps should be abandoned where direct measurement is available.

Preservation of nutrients during long-term storage of source-separated yellowwater.

Source separation of human urine (yellowwater) enhances the sustainability of wastewater management and efficiency of nutrient recovery and recycling. Storage of source-separated yellowwater is recommended prior to agronomic reuse. At this point, it is of immense interest to determine the effect of storage time on quality of yellowwater. Therefore, this study focused on examining changes in some chemical properties of raw, undiluted, freshly collected, source-separated yellowwater stored for a period of 1 year under different temperature regimes: cold (4 °C), mild (10 °C) and warm (22 °C). Chemical parameters (biochemical oxygen demand (BOD(5)), N-tot, N-NO(2), N-NO(3), N-NH(4), P-tot, K, S, and pH), with the main focus on fertiliser nutrient compounds intended for agricultural utilisation, were tested. The outcomes revealed that both nitrification and denitrification processes took place in the stored yellowwater, and an increase in the pH level of up to pH greater than 9 was observed. The study found that the main macronutrients can be well preserved in yellowwater, as there were no substantial changes in the contents of these elements over a 1 year storage period at the three temperatures tested.

Mitochondrial aquaporin-8 in renal proximal tubule cells: evidence for a role in the response to metabolic acidosis.

Mitochondrial ammonia synthesis in proximal tubules and its urinary excretion are key components of the renal response to maintain acid-base balance during metabolic acidosis. Since aquaporin-8 (AQP8) facilitates transport of ammonia and is localized in inner mitochondrial membrane (IMM) of renal proximal cells, we hypothesized that AQP8-facilitated mitochondrial ammonia transport in these cells plays a role in the response to acidosis. We evaluated whether mitochondrial AQP8 (mtAQP8) knockdown by RNA interference is able to impair ammonia excretion in the human renal proximal tubule cell line, HK-2. By RT-PCR and immunoblotting, we found that AQP8 is expressed in these cells and is localized in IMM. HK-2 cells were transfected with short-interfering RNA targeting human AQP8. After 48 h, the levels of mtAQP8 protein decreased by 53% (P < 0.05). mtAQP8 knockdown decreased the rate of ammonia released into culture medium in cells grown at pH 7.4 (-31%, P < 0.05) as well as in cells exposed to acid (-90%, P < 0.05). We also evaluated mtAQP8 protein expression in HK-2 cells exposed to acidic medium. After 48 h, upregulation of mtAQP8 (+74%, P < 0.05) was observed, together with higher ammonia excretion rate (+73%, P < 0.05). In vivo studies in NH(4)Cl-loaded rats showed that mtAQP8 protein expression was also upregulated after 7 days of acidosis in renal cortex (+51%, P < 0.05). These data suggest that mtAQP8 plays an important role in the adaptive response of proximal tubule to acidosis possibly facilitating mitochondrial ammonia transport.

Weight, age and coefficients of variation in renal solute excretion.

BACKGROUND: Homoscedasticity (constant variance over axes or among statistical factors) is an integral assumption of most statistical analyses. However, a number of empirical studies in model organisms and humans demonstrate significant differences in residual variance (that component of phenotype unexplained by known factors) or intra-individual variation among genotypes. Our work suggests that renal traits may be particularly susceptible to randomization by genetic and non-genetic factors, including endogenous variables like age and weight. METHODS: We tested associations between age, weight and intra-individual variation in urinary calcium, citrate, chloride, creatinine, potassium, magnesium, sodium, ammonium, oxalate, phosphorus, sulfate, uric acid and urea nitrogen in 9,024 male and 6,758 female kidney stone patients. Coefficients of variation (CVs) were calculated for each individual for each solute from paired 24-hour urines. Analysis of CVs was corrected for inter-measurement collection variance in creatinine and urine volume. CVs for sodium and urea nitrogen were included to correct for dietary salt and protein. RESULTS: Age was positively associated with individual CVs for calcium and negatively associated with CVs for potassium, ammonium and phosphorus (p(FDR) < 0.01). Weight was associated with CVs for creatinine, magnesium and uric acid, and negatively associated with CVs for calcium, potassium and oxalate (p(FDR) < 0.05). CONCLUSION: Intra-individual variation changes over age and weight axes for numerous urinary solutes. Changing residual variance over age and weight could cause bias in the detection or estimation of genetic or environmental effects. New methodologies may need to account for such residual unpredictability, especially in diverse collections.

Detection of singly- and doubly-charged quaternary ammonium drugs in equine urine by liquid chromatography/tandem mass spectrometry.

Quaternary ammonium drugs (QADs) are anticholinergic agents some of which are known to have been abused or misused in equine sports. A recent review of literature shows that the screening methods reported thus far for QADs mainly cover singly-charged QADs. Doubly-charged QADs are extremely polar substances which are difficult to be extracted and poorly retained on reversed-phase columns. It would be ideal if a comprehensive method can be developed which can detect both singly- and doubly-charged QADs. This paper describes an efficient liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous detection and confirmation of 38 singly- and doubly-charged QADs at sub-parts-per-billion (ppb) to low-ppb levels in equine urine after solid-phase extraction. Quaternary ammonium drugs were extracted from equine urine by solid-phase extraction (SPE) using an ISOLUTE(®) CBA SPE column and analysed by LC/MS/MS in the positive electrospray ionisation mode. Separation of the 38 QADs was achieved on a polar group embedded C18 LC column with a mixture of aqueous ammonium formate (pH 3.0, 10 mM) and acetonitrile as the mobile phase. Detection and confirmation of the 38 QADs at sub-ppb to low-ppb levels in equine urine could be achieved within 16 min using selected reaction monitoring (SRM). Matrix interference of the target transitions at the expected retention times was not observed. Other method validation data, including precision and recovery, were acceptable. The method was successfully applied to the analyses of drug-administration samples.

Laboratory diagnostics in the urine of young and pregnant ewes.

OBJECTIVE: The purpose of this study was to identify values for net acid base excretion (NABE) which are significant indicators of the acid-base equilibrium in pregnant and young ewes and to show its relationship with other parameters (base, acid, ammonium [NH4], base-acid quotient, sodium [Na], potassium [K], calcium [Ca]) in ovine urine. In contrast to dairy cows, data are rare on these parameters in ewes. MATERIAL AND METHODS: A total of 99 animals were used in the study, consisting of 56 young (average of 5.6±1.1 months) and 43 pregnant ewes (average of 35.2±18.8 months). Measurement of fractional NABE in urine samples was carried out according to the method reported by Kutas. The pH value of the urine was measured with a laboratory pH meter. Na, K and total Ca were measured with a flame photometer. RESULTS: For all values except Na significant differences occurred between urine samples of pregnant ewes and young ewes (p<0.001). Base, acid, NH4, NABE, K and Ca values were significantly higher in the urine of the youngs than in pregnant ewes. In young ewes, a strong correlation was found between NABE and base values while a weak correlation could be observed between pH and base values. In pregnant ewes, strong NABE-base, NABE-K, K-acid and K-base correlations were found as well as weak NH4-base, NH4-NABE and NH4-K correlations. There was a strongly positive correlation between NABE and NH4 in pregnant ewes, while a weak negative correlation between those values was observed in young ewes. CONCLUSION: For the first time, we established values for NABE and certain other parameters in urine of pregnant ewes and young ewes. It was shown that the acid-base balance in pregnant ewes and young ewes can be evaluated by measuring NABE and certain trace elements in urine like in cattle.

[Effects of acid and alkaline stress on energy metabolism of Oreochromis niloticus juveniles with different body mass].

A 6 x 3 factorial laboratory experiment was conducted to study the effects of acid and alkaline stress (pH 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0) on the oxygen consumption rate (OR), ammonia excretion rate (NR), and lactate dehydrogenase (LDH) and Na+ -K+ adenosine triphosphatase (Na+ -K+ ATPase) activities of Oreochromis niloticus juveniles with body mass 1.02, 5.13, and 10.31 g. With increasing pH, the juveniles OR and NR increased first, peaked at pH 7.0-8.0, and decreased then. The OR and NR increased with increasing body mass, and their relationships fitted power equations. The linear effects of pH and body mass and the quadratic effect of pH on the OR and NR were highly significant (P < 0.01), but the pH and body mass had less synergistic effect on the OR and NR (P > 0.05). Regressive equations of pH and body mass with the OR and NR were established, the R2 being 0.942 and 0.936, respectively (P < 0.01). Body mass had significant effects on the O:N ratio (P < 0.01), whereas acid and alkaline stress could alter the energy source utilization patterns of the juveniles. High pH was not favorable to the LDH activity, but favorable to the Na+ -K+ ATPase activity. The pH had linear and quadratic effects on the LDH and Na+ -K+ ATPase activities (P < 0.01), body mass had no significant effect on the Na+ -K+ ATPase activity, and the pH and body mass had no synergistic effect on the two enzyme activities (P > 0.05).

Diet-induced metabolic acidosis.

The modern Western-type diet is deficient in fruits and vegetables and contains excessive animal products, generating the accumulation of non-metabolizable anions and a lifespan state of overlooked metabolic acidosis, whose magnitude increases progressively with aging due to the physiological decline in kidney function. In response to this state of diet-derived metabolic acidosis, the kidney implements compensating mechanisms aimed to restore the acid-base balance, such as the removal of the non-metabolizable anions, the conservation of citrate, and the enhancement of kidney ammoniagenesis and urinary excretion of ammonium ions. These adaptive processes lower the urine pH and induce an extensive change in urine composition, including hypocitraturia, hypercalciuria, and nitrogen and phosphate wasting. Low urine pH predisposes to uric acid stone formation. Hypocitraturia and hypercalciuria are risk factors for calcium stone disease. Even a very mild degree of metabolic acidosis induces skeletal muscle resistance to the insulin action and dietary acid load may be an important variable in predicting the metabolic abnormalities and the cardiovascular risk of the general population, the overweight and obese persons, and other patient populations including diabetes and chronic kidney failure. High dietary acid load is more likely to result in diabetes and systemic hypertension and may increase the cardiovascular risk. Results of recent observational studies confirm an association between insulin resistance and metabolic acidosis markers, including low serum bicarbonate, high serum anion gap, hypocitraturia, and low urine pH.

Nitrogen recovery from source-separated human urine using clinoptilolite and preliminary results of its use as fertilizer.

The use of source separated human urine as fertilizer is one of the major suggestions of the new sanitation concept ECOSAN. Urine is rich in nitrogen, phosphorus and potassium which act as plant nutrients, however its salinity is high for agricultural and landscape purposes. Moreover, characteristics change significantly throughout storage where salinity increases to higher values as the predominant form of nitrogen shifts from urea to ammonium. Transferring nitrogen in human urine onto the natural zeolite clinoptilolite and using the subsequently recovered ammonium from the exhausted clinoptilolite for agricultural/landscape purposes is suggested as an indirect route of using urine in this work. Results reporting the outcome of the proposed process together with characterization of fresh and stored urine, and preliminary work on the application of the product on the landscape plant Ficus elastica are presented. Up to 97% of the ammonium in stored urine could be transferred onto clinoptilolite through ion exchange and about 88% could be recovered subsequently from exhausted clinoptilolite, giving an overall recovery of 86%. Another important merit of the suggested process was the successful elimination of salinity. Preliminary experiments with Ficus elastica had shown that the product, i.e. clinoptilolite exhausted with ammonium, was compatible with the synthetic fertilizer tested.

Metabolic basis for low urine pH in type 2 diabetes.

BACKGROUND AND OBJECTIVES: Type 2 diabetes is associated with excessively low urine pH, which increases the risk for uric acid nephrolithiasis. This study was conducted to assess the metabolic basis responsible for the excessive urinary acidity of individuals with type 2 diabetes. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Nine non-stone-forming patients who had type 2 diabetes and low urine pH and 16 age- and body mass index-matched non-stone-forming volunteers without type 2 diabetes were maintained on a constant metabolic diet for 7 days, and 24-hour urine was collected on the last 2 days of the diet. RESULTS: Urine dietary markers (potassium, sulfate, phosphorus, and urea nitrogen) were not different between the two groups. Patients with type 2 diabetes exhibited a significantly lower 24-hour urine pH (5.45+/-0.27 versus 5.90+/-0.42; P<0.01) and higher net acid excretion (NAE; 57+/-12 versus 38+/-18 mEq/d; P<0.01) compared with control subjects. The proportion of NAE excreted as ammonium (NH4+/NAE) was significantly lower in patients with type 2 diabetes than in control subjects (0.70+/-0.12 versus 0.94+/-0.36; P<0.01); however, the greater NAE in patients with type 2 diabetes was not accounted for by the differences in unmeasured urinary anions. CONCLUSIONS: The overly acidic urine in patients with type 2 diabetes persists after controlling for dietary factors, body size, and age. The lower pH is due to a combination of greater NAE and lower use of ammonia buffers in patients with diabetes, which predisposes them to uric acid urolithiasis.

Deletion of the anion exchanger Slc26a4 (pendrin) decreases apical Cl(-)/HCO3(-) exchanger activity and impairs bicarbonate secretion in kidney collecting duct.

The anion exchanger Pendrin, which is encoded by SLC26A4 (human)/Slc26a4 (mouse) gene, is localized on the apical membrane of non-acid-secreting intercalated (IC) cells in the kidney cortical collecting duct (CCD). To examine its role in the mediation of bicarbonate secretion in vivo and the apical Cl(-)/HCO(3)(-) exchanger in the kidney CCD, mice with genetic deletion of pendrin were generated. The mutant mice show the complete absence of pendrin expression in their kidneys as assessed by Northern blot hybridization, Western blot, and immunofluorescence labeling. Pendrin knockout (KO) mice display significantly acidic urine at baseline [pH 5.20 in KO vs. 6.01 in wild type (WT); P < 0.0001] along with elevated serum HCO(3)(-) concentration (27.4 vs. 24 meq/l in KO vs. WT, respectively; P < 0.02), consistent with decreased bicarbonate secretion in vivo. The urine chloride excretion was comparable in WT and KO mice. For functional studies, CCDs were microperfused and IC cells were identified by their ability to trap the pH fluorescent dye BCECF. The apical Cl(-)/HCO(3)(-) exchanger activity in B-IC and non-A, non-B-IC cells, as assessed by intracellular pH monitoring, was significantly reduced in pendrin-null mice. The basolateral Cl(-)/HCO(3)(-) exchanger activity in A-IC cells and in non-A, non-B-IC cells, was not different in pendrin KO mice relative to WT animals. Urine NH(4)(+) (ammonium) excretion increased significantly, consistent with increased trapping of NH(3) in the collecting duct in pendrin KO mice. We conclude that Slc26a4 (pendrin) deletion impairs the secretion of bicarbonate in vivo and reduces apical Cl(-)/HCO(3)(-) exchanger activity in B-IC and non-A, non-B-IC cells in CCD. Additional apical Cl(-)/HCO(3)(-) exchanger(s) is (are) present in the CCD.

Potassium restriction, high protein intake, and metabolic acidosis increase expression of the glutamine transporter SNAT3 (Slc38a3) in mouse kidney.

Kidneys produce ammonium to buffer and excrete acids through metabolism of glutamine. Expression of the glutamine transporter Slc38a3 (SNAT3) increases in kidney during metabolic acidosis (MA), suggesting a role during ammoniagenesis. Potassium depletion and high dietary protein intake are known to elevate renal ammonium excretion. In this study, we examined SNAT3, phosphate-dependent glutaminase (PDG), and phosphoenolpyruvate carboxykinase (PEPCK) regulation during a control (0.36%) or low-K(+) (0.02%) diet for 7 or 14 days or a control (20%) or high-protein (50%) diet for 7 days. MA was induced in control and low-K(+) groups by addition of NH(4)Cl. Urinary ammonium excretion increased during MA, after 14-day K(+) restriction alone, and during high protein intake. SNAT3, PDG, and PEPCK mRNA abundance were elevated during MA and after 14-day K(+) restriction but not during high protein intake. SNAT3 protein abundance was enhanced during MA (both control and low K(+)), after 14-day low-K(+) treatment alone, and during high protein intake. Seven-day dietary K(+) depletion alone had no effect. Immunohistochemistry showed SNAT3 staining in earlier parts of the proximal tubule during 14-day K(+) restriction with and without NH(4)Cl treatment and during high protein intake. In summary, SNAT3, PDG, and PEPCK mRNA expression were congruent with urinary ammonium excretion during MA. Chronic dietary K(+) restriction, high protein intake, and MA enhance ammoniagenesis, an effect that may involve enhanced SNAT3 mRNA and protein expression. Our data suggest that SNAT3 plays an important role as the glutamine uptake mechanism in ammoniagenesis under these conditions.

A role for Rhesus factor Rhcg in renal ammonium excretion and male fertility.

The kidney has an important role in the regulation of acid-base homeostasis. Renal ammonium production and excretion are essential for net acid excretion under basal conditions and during metabolic acidosis. Ammonium is secreted into the urine by the collecting duct, a distal nephron segment where ammonium transport is believed to occur by non-ionic NH(3) diffusion coupled to H(+) secretion. Here we show that this process is largely dependent on the Rhesus factor Rhcg. Mice lacking Rhcg have abnormal urinary acidification due to impaired ammonium excretion on acid loading-a feature of distal renal tubular acidosis. In vitro microperfused collecting ducts of Rhcg(-/-) acid-loaded mice show reduced apical permeability to NH(3) and impaired transepithelial NH(3) transport. Furthermore, Rhcg is localized in epididymal epithelial cells and is required for normal fertility and epididymal fluid pH. We anticipate a critical role for Rhcg in ammonium handling and pH homeostasis both in the kidney and the male reproductive tract.

Low bone density in children with hypercalciuria and/or nephrolithiasis.

The objective of this study was to identify how many children with hypercalciuria and/or nephrolithiasis have a low bone density and whether the risk of low bone density can be identified by 24-h urine stone-risk profiles and/or growth parameters. A retrospective chart review was performed on 110 idiopathic hypercalciuria and/or kidney stone patients who received both a 24-h urine for stone-risk profile and a dual-energy X-ray densitometry scan. Patients were divided into low bone density vs. normal bone density groups and hypercalcuria verus nephrolithiasis groups and analyzed for differences in growth parameters, urine stone-risk profiles, and bone densities. Overall, 47% had a bone density z score < -1, and 26% had a bone density z score < -2. Patients with a low bone density had a higher body mass index and lower urine creatinine and ammonium than those with a normal bone density. Patients with nephrolithiasis had a lower bone density z score than patients with hypercalcuria and no nephrolithiasis. Clinicians should be aware of the increased incidence of low bone density in children with hypercalciuria and nephrolithiasis. The effect of hypercalciuria and nephrolithiasis treatment on bone density and the natural progression of the bone density in the studied patient population warrants further investigation.