Changes In Urinary Excretion Research Articles

Effects of two- and twelve-weeks sodium-glucose cotransporter 2 inhibition on DNA and RNA oxidation: two randomized, placebo-controlled trials

Animal studies have shown that SGLT2 inhibition decreases oxidative stress, which may explain the cardiovascular protective effects observed following SGLT2 inhibition treatment. Thus, we investigated the effects of two and twelve weeks SGLT2 inhibition on DNA and RNA oxidation. Individuals with type 2 diabetes (n = 31) were randomized to two weeks of treatment with the SGLT2 inhibitor empagliflozin treatment (25 mg once daily) or placebo. The primary outcome was changes in DNA and RNA oxidation measured as urinary excretion of 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo), respectively. In another trial, individuals with type 2 diabetes (n = 35) were randomized to twelve weeks of dapagliflozin treatment (10 mg once daily) or placebo in a crossover study. Changes in urinary excretion of 8-oxodG and 8-oxoGuo were investigated as a posthoc analysis. Compared with placebo treatment, two weeks of empagliflozin treatment did not change urinary excretion of 8-oxodG (between-group difference: 0.3 nmol/24-hour (95% CI: −4.2 to 4.8)) or 8-oxoGuo (1.3 nmol/24-hour (95% CI: −4.7 to 7.3)). From a mean baseline 8-oxodG/creatinine urinary excretion of 1.34 nmol/mmol, dapagliflozin-treated individuals changed 8-oxodG/creatinine by −0.17 nmol/mmol (95% CI: −0.29 to −0.04) following twelve weeks of treatment, whereas placebo-treated individuals did not change 8-oxodG/creatinine (within-group effect: 0.10 nmol/mmol (95% CI: −0.02 to 0.22)) resulting in a significant between-group difference (p = 0.01). Urinary excretion of 8-oxoGuo was unaffected by dapagliflozin treatment. In conclusion, two weeks of empagliflozin treatment did not change DNA or RNA oxidation. However, a posthoc analysis revealed that longer-term dapagliflozin treatment decreased DNA oxidation. Clinicaltrials.gov: NCT02890745 and NCT02914691. Highlights Plasma ferritin correlated with DNA and RNA oxidation in individuals with T2D. Twelve weeks dapagliflozin treatment decreased DNA oxidation. Dapagliflozin and empagliflozin treatment did not change RNA oxidation. Lipid peroxidation was unaffected by two weeks empagliflozin treatment.

Profiling of Urinary Glucuronidated Bile Acids across Age Groups.

We investigated the age-dependent changes in urinary excretion of glucuronidated bile acids at the C-3 position. Bile acid 3-glucuronides accounted for 0.5% of urinary bile acids in neonates, and the proportion of bile acid 3-glucuronides plateaued at 1-3 years of age. The 3-glucuronides of secondary bile acids were first secreted at 3 months of age, the same time as the establishment of the gut bacterial flora in infants. A considerable portion of bile acid 3-glucuronides were present as non-amidated forms. Our results indicate dynamic hepatic enzyme activity in which the levels of uridine 5'-diphospho-glucuronosyltransferases (UGTs) differ by age group, with higher glucuronidation activity of UGTs towards nonamidated bile acids than amidated bile acids.

RF21 | PSAT99 Urinary adrenal steroidome modifications by Ketoconazole and Metyrapone alter the urinary free cortisol immunoassay reliability in Cushing Syndrome

Abstract Introduction Twenty-four-hour urinary free cortisol (24h-UFC) is the most used test for follow-up decision-making in patients with Cushing syndrome (CS) under adrenal-directed medical treatment. In CS patients without medical treatment, 24h-UFC determinations by immunoassays (IA) are commonly overestimated because of interference by cross-reacting steroid metabolites. However, it is unknown how induced changes by Ketoconazole (KTZ) or Metyrapone (MTP) on the excretion of urinary steroid metabolites alter the IA cross-reactivity interference of 24h-UFC determinations and what are its clinical implications. Methods 24h-UFC was analyzed by IA and gas chromatography-mass spectrometry (GC-MS) in 193 samples (81 before treatment, 73 during KTZ and 39 during MTP) from 34 CS patients. In addition, 35 urinary steroids were analyzed by GC-MS on each patient before and during treatment. Results Before treatment, 24h-UFC*IA determinations were overestimated by a factor of 1.75: 1 (95% CI 1.60 - 1.94) compared to those by GC-MS. During treatment with KTZ, the cross-reactivity interference of the 24h-UFC results by IA decreased in a dose-dependent manner (p = 0.000). Thus, in patients taking KTZ >400mg/day, 24h-UFC*IA determinations were no longer overestimated, giving similar results to those by GC-MS (factor of 0.98: 1, 95% CI, 0.83–1.20). Baseline 24h-UFC and upper limit normal (ULN) reductions were magnified (>25%) when assessed by IA vs GC-MS leading to higher falsely KTZ efficacy and misleading biochemical classification of 15% of patients. Despite dosage, in patients taking MTP the IA cross-reactivity only decreased 0.55 which resulted in persistence of overestimated 24h-UFC results (factor of 1.33: 1, 95% CI, 1.09 -1.76). When comparing MTP efficacy among IA vs GC-MS results, ULN baseline reductions were >50% different in some patients, while in others, similar reductions were found. Acceptable 24h-UFC method agreement between GC-MS and IA before treatment (R2 = 0.954) declined in patients under KTZ (R2 = 0.632) but not in MTP (R2 = 0.917). Urinary excretion changes of 22 urinary steroid metabolites explained 86% of the 24h-UFC*IA interference. 6β-Hydroxy-cortisol (6β-OH-cortisol) was the metabolite determining most of the 24h-UFC cross-reactivity variability (R2= 48.3%, p = 0.000) followed by 20α-dihydrocortisol (20α-DHF) (R2= 24.4%, p = 0.000). Each treatment induced a unique urinary steroid signature (p <0.001) and larger excretion reductions of 6β-OH-cortisol, 20α-DHF and 18-Hydroxy-cortisol in patients with KTZ elucidated the higher decrement of interference in the 24h-UFC by IA, compared to MTP treated patients (p = 0.001). Conclusion KTZ and MTP alter distinctly the urinary excretion of IA cross-reactive steroid metabolites, thus decreasing the cross-reactive interference of 24h-UFC*IA determinations present before treatment in a unique manner. Consequently, this interference reduction in 24h-UFC*IA leads to loss of method agreement with GC-MS and high risk of overestimating the biochemical impact of KTZ and MTP in controlling CS because of poor reliability of ULN. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m., Sunday, June 12, 2022 12:36 p.m. - 12:41 p.m.

Ketoconazole- and Metyrapone-Induced Reductions on Urinary Steroid Metabolites Alter the Urinary Free Cortisol Immunoassay Reliability in Cushing Syndrome.

IntroductionTwenty-four-hour urinary free cortisol (24h-UFC) is the most used test for follow-up decision-making in patients with Cushing syndrome (CS) under medical treatment. However, 24h-UFC determinations by immunoassays (IA) are commonly overestimated because of steroid metabolites’ cross-reaction. It is still uncertain how ketoconazole (KTZ)- and metyrapone (MTP)-induced changes on the urinary steroid metabolites can alter the 24h-UFC*IA determinations’ reliability.Methods24h-UFC was analyzed by IA and gas chromatography-mass spectrometry (GC-MS) in 193 samples (81 before treatment, 73 during KTZ, and 39 during MTP) from 34 CS patients. In addition, urinary steroidome was analyzed by GC-MS on each patient before and during treatment.ResultsBefore treatment, 24h-UFC*IA determinations were overestimated by a factor of 1.75 (95% CI 1.60–1.94) compared to those by GC-MS. However, during KTZ treatment, 24h-UFC*IA results were similar (0.98:1) to those by GC-MS (95% CI, 0.83–1.20). In patients taking MTP, IA bias only decreased 0.55, resulting in persistence of an overestimation factor of 1.33:1 (95% CI, 1.09–1.76). High method agreement between GC-MS and IA before treatment (R2 = 0.954) declined in patients under KTZ (R2 = 0.632) but not in MTP (R2 = 0.917). Upper limit normal (ULN) reductions in patients taking KTZ were 27% larger when using 24h-UFC*IA compared to 24h-UFC*GC-MS, which resulted in higher false efficacy and misleading biochemical classification of 15% of patients. Urinary excretion changes of 22 urinary steroid metabolites explained 86% of the 24h-UFC*IA interference. Larger urinary excretion reductions of 6β-hydroxy-cortisol, 20α-dihydrocortisol, and 18-hydroxy-cortisol in patients with KTZ elucidated the higher 24h-UFC*IA bias decrement compared to MTP-treated patients.ConclusionKTZ and MTP alter the urinary excretion of IA cross-reactive steroid metabolites, thus decreasing the cross-reactive interference of 24h-UFC*IA determinations present before treatment. Consequently, this interference reduction in 24h-UFC*IA leads to loss of method agreement with GC-MS and high risk of overestimating the biochemical impact of KTZ and MTP in controlling CS because of poor reliability of reference ranges and ULN.

Neonatal Proteinuria in Calves-A Quantitative Approach.

Simple SummaryA newborn’s survival depends on the quick adaptation of the organism to new environmental conditions. Newborn calves show high somatic maturity compared to, for example, human newborns, but their body functions with a lower efficiency than that of adult cattle. Adaptation processes concern all organs, including the kidneys, which are not morphologically mature after birth. The ongoing morphological changes imply functional alterations in the kidneys. There is an increase in blood flow through the kidneys and the glomerular filtration rate, as well as an increase in the efficiency of resorption processes of primary urine components into the blood, including proteins. Protein is present in trace amounts in the urine of healthy adults of various species. It can occur in larger amounts in the urine of sick animals, as well as in certain physiological states, e.g., in newborns. The presence of protein in the urine of newborns in the quantity exceeding 300 mg/24 h/m2 is called neonatal proteinuria. The causes of proteinuria in healthy newborns have not been clearly elucidated. Many studies have focused on proteinuria in newborns and sick children and sick animals, especially dogs and cats. The present study was the first to quantify the hypothesis of the occurrence of proteinuria in healthy calves in the first week of life and to assess its intensity and dynamics, based on the analysis of changes in total protein excretion in the urine and its fractions differing in molecular weight (albumin, low molecular weight proteins (LMW) and high molecular weight proteins (HMW)). It should be noted that the analysis of excreted protein fractions is a sensitive diagnostic indicator of the type of kidney disease, e.g., increased HMW protein excretion may indicate damage to the renal glomeruli, and loss of LMW proteins may indicate renal tubular disease.Urine testing is a convenient, non-invasive method of obtaining information about body functions. Depending on the intended purpose, urine testing may be qualitative and/or quantitative. Urine analysis can also include proteins. There are no data in the literature on the occurrence of proteinuria in healthy neonatal calves. The present study was the first that aimed to quantify the hypothesis of proteinuria occurrence in these animals in the first week of life, to assess its intensity and dynamics and to understand the underlying causes of proteinuria in healthy calves. The research was carried out on 15 healthy calves in the first seven days of life. Calves were catheterized to determine minute diuresis. Total protein concentration was determined in blood plasma and urine. Urine proteins were separated by electrophoresis (SDSPAGE) and their concentration and percentage were determined by densitometry using an image archiving and analysis software. The separated proteins were divided into three groups according to molecular weight for albumin, LMW and HMW proteins. The results were standardized per 1 m2 of body surface area and statistically analyzed. Neonatal proteinuria was demonstrated in healthy calves, mainly resulting from the high concentration of LMW proteins in the urine. Their percentages decreased significantly from 84.46% on the first day of calves’ life to 64.02% on day 7. At the same time, a statistically significant increase was observed in the proportion of albumin and high molecular weight proteins in urine total protein. Albumin percentage increased from 9.54% (on day 1) to almost 20% (on day 7), while the proportion of HMW proteins increased from 6.68% to 18.13%, respectively. The concentration of total protein in the urine of newborn calves amounted to 14.64 g/L and decreased statistically significantly during the first 72 h of postnatal life, stabilizing at the level of 3–4 g/L. The mean value of total protein excretion in the first week of life was 4.81 mg/min/m2 (i.e., 6.93 g/24 h/m2). The analysis of protein concentration in the urine and its excretion, as well as changes in urinary excretion of the tested protein fractions, indicated that neonatal proteinuria in healthy neonatal calves was tubular (i.e., main reason is the reduced absorption of proteins in nephrons). In addition, research showed that there was a rapid improvement in resorptive mechanisms in tubular cells. It should be assumed that the filtration barrier in the kidneys of these animals after birth is morphologically prepared to retain high molecular weight proteins. It seems that the increased permeability of the filtration barrier in the glomeruli does not necessarily indicate the immaturity of the kidneys, but may indicate the kidneys’ adaptation to excess protein removal from the body during feeding with high-protein food (colostrum), with an open intestinal barrier enabling protein absorption from the gastrointestinal tract to the blood.

Abstract LB221: Randomized crossover trial evaluating detoxication of tobacco carcinogens by broccoli seed and sprout extract in heavy smokers

Abstract Randomized crossover trial evaluating detoxication of tobacco carcinogens by broccoli seed and sprout extract in heavy smokersJulie E. Bauman, Chiu-Hsieh Hsu, Sara Centuori, Jose Guillen-Rodriguez, Linda Garland, Emily Ho, Lisa Bengtson, Malgorzata Wojtowicz, Eva Szabo, H-H Sherry ChowIntroduction: Diets high in cruciferous vegetables are associated with reduced risk of tobacco-related cancers. Crucifers are rich in the phytochemical glucoraphanin (GR), which is hydrolyzed by myrosinase to its bioactive form, sulforaphane (SF). SF upregulates the NRF2 transcription factor and downstream target genes in the antioxidant response element. As GR is concentrated in broccoli seeds relative to mature plants, broccoli seed preparations (BSP) are under development as chemopreventive agents. BSP increased detoxication of air pollutants including benzene in Qidong, China. Methods: We conducted a randomized crossover trial evaluating the detoxication of benzene and other tobacco carcinogens by the BSP Avmacol, tablets comprised of broccoli seed powder and broccoli sprout extract, in otherwise healthy smokers (≥ 20 pack-years). Each subject was treated with low and high dose BSP (70 vs. 140 GR equivalents daily for 2 weeks), separated by a 2-week washout, with randomization to low-high vs. high-low sequence. The primary endpoint was detoxication of benzene, measured by change in urinary excretion of its mercapturic acid metabolite (S-phenyl mercapturic acid, SPMA). Secondary endpoints included detoxication of the carcinogens acrolein and crotonaldehyde, and SF bioavailability assessed by urinary SF metabolites.Results: 49 subjects were randomized from Feb 2018-Nov 2019: 26 female, mean age 56.3. Treatment-related adverse events (AE) were gastrointestinal; most common were grade 1-2 bloating/cramping/abdominal pain (11; 22%), grade 1 diarrhea (11; 22%), grade 1 flatulence (10; 20%). No grade ≥ 3 AE were observed. One subject withdrew after unrelated AE. Compliance with BSP and biomarker measurements was 98%. Primary and secondary endpoints are presented in the Table. Conclusion: The BSP Avmacol was bioavailable as SF metabolites and significantly increased the acute detoxication products of benzene and acrolein in heavy smokers. Dose LevelChange in Metabolite (IQR)a; N=48P-ValueΔ SF MetabolitesbLow Dose19.93 (6.50, 49.00) nmol/mg Cr<0.0001High Dose40.75 (13.61, 91.89) nmol/mg Cr<0.0001Δ Benzene (SPMA)bLow Dose0.59 (-0.79, 4.65) pmol/mg Cr0.03High Dose0.84 (-1.30, 3.39) pmol/mg Cr0.06Δ Acrolein (3-hydroxypropyl mercapturic acid, 3-HPMA)bLow Dose418.05 (-1943.97, 2268.00) pmol/mg Cr0.47High Dose2250.31 (-1675.74, 7723.57) pmol/mg Cr0.01Δ Crotonaldehyde (3-hydroxy-1-methylpropyl mercapturic acid, 3-HMPMA)bLow Dose-837.57 (-3716.93, 4903.17) pmol/mg Cr0.83High Dose981.94 (-1710.04, 4621.08) pmol/mg Cr0.08aChange: End of Treatment – Baseline; IQR: Interquartile RangebSF metabolites, SPMA, 3-HPMA, and 3-HMPMA were normalized to urine creatinine Citation Format: Julie E. Bauman, Chiu-Hsieh Hsu, Sara Centuori, Jose Guillen-Rodriguez, Linda Garland, Emily Ho, Lisa Bengtson, Malgorzata Wojtowicz, Eva Szabo, H-H Sherry Chow. Randomized crossover trial evaluating detoxication of tobacco carcinogens by broccoli seed and sprout extract in heavy smokers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB221.

Metabolism of testosterone during weight loss in men with obesity

ObjectiveMen with obesity often have low total and, with increasing adiposity, also low free testosterone (T) levels, which can partially restore during weight loss. Although this is partly explained by lower sex hormone binding globulin (SHBG) production and hypothalamic-pituitary downregulation, it is still not unravelled whether changes in androgen metabolism contribute to this phenomenon. Therefore, early changes in urinary excretion of T and its metabolites, during weight loss, in men with obesity are investigated. DesignLongitudinal study. MethodsFourteen men with obesity (age 52(45–60)years, BMI 42.6(41.8–44.8)kg/m²) underwent gastric bypass surgery (GBS). Before surgery and 3 weeks, 6 weeks, 6 months and 1 year thereafter, 24 h urine and fasting serum samples were collected. Serum T and estradiol (E2) levels were analyzed using LC-MS/MS and urinary metabolites of T with GC-MS/MS. ResultsAlready three weeks after GBS, serum SHBG and total T levels increased and remained increased as compared to baseline (all,p < 0.0125). Gonadotropins and (free) E2 levels were unchanged, serum E2/T ratio decreased (p < 0.0125). Total amount of urinary T increased non-significantly with mean increases of 53 % one year after GBS (p = 0.026). Urinary E2/T, estrone/T, 3α-androstanediol/T and androsterone/T ratios decreased after GBS (p < 0.0125). ConclusionsRestoration of circulating T levels during weight loss in this population is not only brought about by normalization of circulating SHBG levels, but increased production of and alterations in T metabolism also contribute. More specifically, relative decreases in aromatization and lower 5α-reductase activity might also be involved in restoring T levels in men with obesity.

Oral administration of Costa Rican guava (Psidium friedrichsthalianum) juice induces changes in urinary excretion of energy-related compounds in Wistar rats determined by 1H NMR

IntroductionPsidium friedrichsthalianum fruits have in vitro antioxidant activity and their intake has been suggested to have potential health benefits; however, there is still no evidence on the potential in vivo effect of their consumption. ObjectiveWe aim to study the effect of the oral administration of a P. friedrichsthalianum juice on the urinary metabolome of adult male Wistar rats. MethodsAcute and short-term interventions were carried out and urine sample metabolites were analyzed in a 600 MHz NMR instrument with a NOESY-presat sequence.and post-treatment effects were identified by comparison against control conditions. ResultsAcute administration caused a decrease in the excretion of citrate and allantoin in the 0 to 6 h period and citrate and α-ketoglutarate in the 6 to 12 h period. Lactate and Krebs cycle intermediates showed reduced excretion in the 12 to 24 h period. Short-term administration (for 7 days) decreased the excretion of Krebs cycle intermediates together with lactate and allantoin. The effect was stronger after short-term administration suggesting a dose-dependent effect. ConclusionP. friedrichsthalianum juice caused a change in rat excretion of energy metabolites, probably associated with an increase in aerobic metabolism, suggesting in vivo modulation of the redox balance.

Differential time responses in inflammatory and oxidative stress markers after a marathon: An observational study

ABSTRACT Acute and adaptive changes in systemic markers of oxidatively generated nucleic acid modifications (i.e., 8-oxo-7,8-dihydro-2ʹ-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo)) as well as inflammatory cytokines (i.e., C-reactive protein, interleukin-6, interleukin-10, and tumour necrosis factor alpha), a liver hormone (i.e., fibroblast growth factor 21 (FGF21)), and bone metabolism markers (sclerostin, osteocalcin, C-terminal telopeptide, and N-terminal propeptide of type 1 procollagen) were investigated following a marathon in 20 study participants. Immediate changes were observed in inflammatory cytokines, FGF21, and bone metabolism markers following the marathon. In contrast, no immediate changes in urinary excretion of 8-oxodG and 8-oxoGuo were evident. Four days after the marathon, decreased urinary excretion of 8-oxodG (-2.9 (95% CI -4.8;-1.1) nmol/24 h, P < 0.01) and 8-oxoGuo (-5.8 (95% CI -10.3;-1.3) nmol/24 h, P = 0.02) was observed. The excretion rate of 8-oxodG remained decreased 7 days after the marathon compared to baseline (-2.3 (95%CI -4.3;-0.4) nmol/24 h, P = 0.02), whereas the excretion rate of 8-oxoGuo was normalized. In conclusion marathon participation immediately induced a considerable inflammatory response, but did not increase excretion rates of oxidatively generated nucleic acid modifications. In fact, a delayed decrease in oxidatively generated nucleic acid modifications was observed suggesting adaptive antioxidative effects following exercise. Abbreviations 8-oxodG: 8-oxo-7,8-dihydro-2ʹ-deoxyguanosine; 8-oxoGuo: 8-oxo-7,8-dihydroguanosine; CI: confidence interval; CTX: C-terminal telopeptide of type 1 collagen; DXA: dual-energy X-ray absorptiometry; ELISA: enzyme-linked immunosorbent assay; FGF21: Fibroblast growth factor 21; h: hour; hsCRP: high sensitivity C-reactive protein; IL: interleukin; IQR: interquartile range; MS: mass spectrometry: P1NP: N-terminal propeptide of type 1 procollagen; TNFα: tumour necrosis factor alpha; UPLC: ultra-performance liquid chromatography

Moderate Consumption of Freeze-dried Blueberry Powder Increased Net Bone Calcium Retention in Healthy Postmenopausal Women: A Randomized Crossover Trial

Abstract Objectives Preclinical studies suggest that blueberry consumption is associated with improved bone health. This study quantified changes in urinary excretion of the long-lived calcium radioisotope 41Ca to assess the dose-response effect of blueberries on bone loss in postmenopausal women. We hypothesized that blueberry consumption would reduce bone loss in a dose-dependent manner. Methods Healthy women at least 4 years past menopause were dosed with 50 nCi of 41Ca and completed a 5-mo equilibration period for 41Ca deposition in bone followed by a 6-wk control period. Subsequently, participants were assigned to a random sequence of 3 intervention periods, each corresponding to a low (17.5 g/d), medium (35 g/d), or high (70 g/d) dose of freeze-dried blueberry powder equivalent to 0.75, 1.5, or 3 cups of fresh blueberries. The blueberry powder was incorporated into granola bars, spread, and drinks consumed daily for 6 wk, followed by a 6-wk washout period. Urinary 41Ca: Ca ratio was measured by accelerator mass spectrometry in 24-h urine collected weekly during intervention and every 3 wk during washout. Serum 25-hydroxyvitamin D and calcium concentrations were assessed prior to each intervention. Serum bone resorption biomarkers and urinary polyphenols were measured at the end of each control and intervention period. Data were analyzed using a linear mixed model and repeated measures ANOVA. Results Fourteen healthy, non-osteoporotic (mean bone mineral density t-score: −1.33) women completed the study. Net bone calcium retention increased by 6% in the low (P &amp;lt; 0.01) and 4% in the medium (P &amp;lt; 0.05) dose intervention compared with the control and washout periods. The high dose had no significant effect on net bone calcium retention (P = 0.19). Urinary excretion of hippuric acid increased dose-dependently with blueberry consumption. No significant relationships were found between bone resorption biomarkers, 25-hydroxyvitamin D, or calcium and any of the interventions. Conclusions A hormetic response was observed to blueberry intake, whereby daily consumption of foods containing 17.5–35 g, but not 70 g, of freeze-dried blueberry powder increased net bone calcium retention. Moderate consumption of blueberries may be an effective strategy to attenuate bone loss in healthy postmenopausal women. Funding Sources NIH/NCCIH, Project Development Team within the ICTSI NIH/NCRR.

Role of the NO/cGMP pathway and renin-angiotensin system in the hypotensive and diuretic effects of aqueous soluble fraction from Crataegus songarica K. Koch.

Fruits of Crataegus songarica K. Koch. (Rosaceae) are commonly used in folk medicine for their diuretic properties to treat hypertension and congestive heart failure. To date, no scientific data has been published to support the diuretic potential. The purpose of this study was to evaluate efficacy and mechanism underlying the hypotensive and diuretic action of C. songarica in normotensive rats and to determine the constituents from the extracts by LC-DAD-MS. Firstly, phytochemical profiling and antioxidant potential of C. songarica extracts was determined. Then, to evaluate changes in blood pressure, different groups of anesthetized normotensive rats were intravenously treated with crude extract (CS-Cr, 10-80mg/kg), aqueous soluble (AS-CS, 0.1-20mg/kg), and n-butanol soluble fractions of C. songarica (BS-CS, 1-80mg/kg). The diuretic effects of CS-Cr (100-500mg/kg, p.o), AS-CS (100-300mg/kg, p.o) and BS-CS (100-300mg/kg, p.o) were evaluated in comparison with hydrochlorothiazide (HCTZ, 10mg/kg, p.o). The urinary volume, sodium, potassium and pH were estimated in the sample collected for 6h from saline-loaded rats. Using pharmacological antagonists or inhibitors, we determine the involvement of acetylcholine, prostaglandins, and nitric oxide in C. songarica induced hypotensive and diuresis action. In addition, the activities of angiotensin converting enzyme, erythrocytary carbonic anhydrase and renal Na+/K+/ATPase were evaluated in vitro. From the LC-DAD-MS analyses, thirty-nine compounds were detected, showing a complex chemical profile and an expressive antioxidant activity "in vitro". Acute treatment with CS-Cr, AS-CS, and BS-CS exhibited significant hypotensive and diuretic potential in normotensive rats. However, AS-CS produced most potent and significant dose-dependent hypotension in normotensive rats, and also produced highly significant diuretic and saluretic effects. Despite the changes in urinary excretion of electrolytes, the plasmatic levels of sodium and potassium were not changed. Previous treatment with atropine and L-NAME significantly reduced the hypotensive and diuretic action of AS-CS in normotensive rats. Moreover, the 7-day treatment with AS-CS also resulted in significant ACE inhibitory activity. This research supports and extends the ethnomedicinal use of C. songarica as diuretic and hypotensive agent. The results showed that AS-CS from C. songarica could present compounds responsible for hypotensive and diuretic activities with no signs of toxicity, and these effects could involve nitric oxide pathway activated by muscarinic receptors or/and inhibition of angiotensin converting enzyme.

Changes in urinary excretion of phthalates, phthalate substitutes, bisphenols and other polychlorinated and phenolic substances in young Danish men; 2009-2017.

During the past two decades human exposure to bisphenol A (BPA) and phthalates such as di-iso-butyl phthalate (DiBP), di-n-butyl phthalate (DnBP), butylbenzyl phthalate (BBzP) and di-(2-ethyl-hexyl) phthalate (DEHP) has received substantial interest due to widespread population exposures and potential endocrine disrupting effects. Therefore, these chemicals have gradually been restricted and phased out through legislation. However, humans are still exposed to a wide range of other less studied phthalates, phthalate substitutes and BPA analogues as well as other polychlorinated and phenolic substances. In this study, we investigated human exposure to these chemicals over the past decade. Three hundred urine samples collected in 2009, 2013 and 2017 (100 samples each year) from young Danish men of the general population, participating in a large on-going cross-sectional study, were selected for the present time trend study. The urinary concentration of metabolites of 15 phthalates, di-2-ethylhexyl terephthalate (DEHTP) and di-iso-nonyl-cyclohexane-1,2-dicarboxylate (DINCH), seven bisphenols including BPA, bisphenol S (BPS) and bisphenol F (BPF), as well as triclosan, triclocarban, benzophenone-3, three chlorophenols and two phenylphenols were analyzed by two new sensitive LC-MS/MS methods developed and validated for the present study. A significant decrease in urinary concentrations over time was observed for the majority of the chemicals. Median concentrations of BPA and the metabolites of DiBP, DnBP, BBzP and DEHP were more than halved from 2009 to 2017. Similar decreases were observed for triclosan and the chloro- and phenylphenols. In contrast, metabolites of the two phthalate substitutes DEHTP and DINCH increased more than 20 and 2 times, respectively. The potential BPA substitutes; BPS and BPF also increased, but only slightly. Despite these new exposure patterns, the exposure to the old well-known chemicals, such as DiBP, DnBP, BBzP, DEHP and BPA was still higher in 2017 compared to the exposure level of the new substitutes such as DEHTP, DINCH, BPS and BPF. A significant decrease in internal exposure to most of the common phthalates and BPA over the past decade was observed, reflecting market changes and regulatory measures implemented in EU. Despite increasing exposures to some of the known phthalate substitutes and BPA analogues, the total amount of each measured chemical group (original and substitute analytes combined) was lower in the more recently collected samples. This indicates only partial direct substitution or substitution by chemicals not covered in this approach, or a general decline in the exposure to these chemical/product groups over the last decade.

Post-transplant laronidase augmentation for children with Hurler syndrome: biochemical outcomes

Allogeneic hematopoietic cell transplantation (HCT) benefits children with Hurler syndrome (MPS-IH). However, survivors remain burdened by substantial MPS-IH related residual disease. We studied the feasibility, safety and biochemical impact of augmentative recombinant intravenous enzyme replacement therapy (IV-ERT) post transplantation. Ten children with MPS-IH and ≥2 years from successful HCT underwent IV-ERT for 2 years’ duration. Patients were monitored for anti-drug antibody (ADA) development, including inhibitory capacity and changes in urinary excretion of glycosaminoglycans (uGAG). Three patients demonstrated low-level ADA at baseline, though all children tolerated IV-ERT well. Eight patients developed ADA over the 2-year study, with 3 (38%) meeting criteria for an inhibitory ADA response. The aggregate cohort experienced a reduction in uGAG from baseline to study end, which was enhanced in children with low or no ADA response. Conversely, children with inhibitory ADA showed increase in uGAG over time. IV-ERT in previously transplanted children with MPS-IH appears safe and can reduce uGAG, although this is reversed by the presence of inhibitory ADA. These data show a biochemical change after initiation of post-HCT IV-ERT, but the occurrence of ADA and inhibitory antibodies are a concern and should be monitored in future efficacy trials. This trial was registered at www.clinicaltrials.gov, NCT01173016, 07/30/2010.

Effects of work schedule and period of exposure on changes in urinary chromium and nickel excretion among rotating shift workers in a stainless-steel plant

ABSTRACTWe investigated the association between the period of exposure and changes in urinary excretion of chromium and nickel among rotating shift workers in a stainless-steel plant. The study participants were composed of two groups: the workers who were occupationally exposed to metals (“exposed group”) and those who were not occupationally exposed to metals (“unexposed group”). The exposed and unexposed groups consisted of 56 and 40 male rotating shift workers, respectively. Urine samples were collected immediately before and immediately after the day shift, evening shift, and night shift. Urinary chromium and nickel were measured using inductively coupled plasma mass spectrometry. To correct for variations in urine dilution, urinary metal concentrations were expressed as a ratio to urinary creatinine concentration. In the exposed group, post-shift urinary excretion of chromium was significantly higher than pre-shift excretion. However, although urinary chromium excretion clearly increased after the day and night shift [63% (p < .0001) and 87% (p < .0001), respectively], urinary chromium excretion after the evening shift was only slightly higher than that measured before the evening shift (8%, p = .028). Similar patterns were found for urinary nickel excretion (p = .0001, 0.20, and 0.18 for the day, evening, and night shifts, respectively). Non-uniform urinary excretion of metals between the day shift, evening shift, and night shift were observed in the exposed group; specifically, urinary metal excretion increased only slightly during the evening shift. In the unexposed group, no significant increase or decrease was found in median urinary chromium or nickel excretion (p= .63–0.87). Work shift-specific permissible exposure level would be necessary.

Camelina sativa Oil, Fatty Fish, and Lean Fish Do Not Markedly Affect Urinary Prostanoids in Subjects with Impaired Glucose Metabolism.

Dietary fatty acids are suggested to affect oxidative stress; however, results from interventions have been inconclusive. The aim was to examine if fatty fish, lean fish, and Camelina sativa oil (CSO) affect the urinary prostanoid levels in subjects with impaired glucose metabolism. Altogether 79 participants aged 43-72 years completed a randomized controlled study lasting 12 weeks. There were four parallel groups, fatty fish, lean fish (four fish meals/week in both), CSO providing 10 g/day alpha-linolenic acid (ALA), and control diet with limited fish and ALA containing oil consumption. Urinary prostanoids (prostaglandin F2α , 5-F2t -isoprostanes and 15-F2t -isoprostane metabolites, isofuran, 8-F3t -isoprostanes, and 4-(RS)-4-F4t -neuroprostane) of 72 participants (age: mean (±SD) 58.9 ± 6.5 years; body mass index: 29.3 ± 2.5 kg/m2 ) collected over 12-h were measured using liquid chromatography tandem-mass spectrometry. Plasma phospholipid fatty acids were determined using gas chromatography. Our study showed that the proportion of ALA in plasma phospholipids increased in the CSO group (overall difference among the groups p-value <0.001). In the fatty fish group, proportions of eicosapentaenoic and docosahexaenoic acids increased (overall p-value <0.001 for both). Prostaglandin F2α was higher in the CSO group than in the control group (p < 0.05), however, there were no other significant changes in urinary excretion of other prostanoids among the study groups. At baseline, arachidonic acid in plasma phospholipids was positively (r = 0.247, p < 0.05) and ALA negatively (r = -0.326, p < 0.05) associated with urinary total isoprostanes. In conclusion, CSO, fatty fish, and lean fish consumption do not cause major changes in oxidative stress markers in subjects with impaired glucose tolerance.

Renal Adaptive Changes in Response to Chronic Metabolic or Respiratory Acidosis: Regulation of Expression of Acid‐base Transporters and Enzymes

Kidneys excrete acid loads mainly by generating and excreting total ammonia (NH3plus NH4+) and to a lesser extent titratable acids (TA). Urinary excretion of NH3/NH4+ and TAincreases significantly in acidosis. We made the assumption that this augmented urinary acid excretion (NH3/NH4+and TA) will differ depending on the type of acid‐load. To test this hypothesis, we conducted in‐vivo experiments on mice to measure urinary NH3/NH4+and TA under metabolic or respiratory acid loads and investigated expression of carbonic anhydrase and acid‐base transporters by Western analysis.We induced metabolic acidosis in 2 groups of mice: one group received an acid diet (AD) supplemented with 0.4N HCl (at 1mg/g of chow); a second group received drinking water fortified with NH4Cl (0.28M) and 1% sucrose (NH4+‐W); a control group received normal diet (ND). Our data showed that urinary NH4+(u‐NH4+) increased gradually in mice on AD or NH4–W diet compared to control (ND) and the increase in u‐NH4+was proportionally higher than that of TA. Unexpectedly, expression of the NH3/NH4+transporters, Rhbg and Rhcg, in both groups of acid‐loaded mice was not significantly different compared to mice on ND. An interesting finding, however, was that expression of CA‐IV was reduced in both acidosis groups. In mice on AD, RNAseq data showed that carbonic anhydrase‐II (CA‐II) was upregulated whereas pendrin (a Cl‐HCO3−exchanger) expression was downregulated.We induced chronic respiratory acidosis by placing mice for 3 days in special chambers where breathing gas mixtures were 8% CO2(balance 21% O2&amp; 71% N2). A control group breathed normal air. There were no significant changes in urinary excretion of TA in both hypercapnic or control groups. However, urinary NH4+in hypercapnic mice increased significantly after 24 hours and continued to gradually increase in days 2 and 3. In control mice, there was a small increase in urinary NH4+excretion in day 1 (p &lt; 0.05), probably caused by housing distress in metabolic cages, but leveled off in days 2 and 3. Western analysis showed that hypercapnia induced a marked increase in expression of Rhbg but only a moderate increase of Rhcg. Chronic hypercapnia increased expression of CA‐IV significantly but decreased expression of pendrin.In summary, we demonstrated that renal response to acidosis varies with the type of acid load. High CO2 acidosis downregulated pendrin even though plasma HCO3−is high. In metabolic acidosis, the increase in urinary NH4+is likely caused by increased ammoniagenesis, whereas upregulation of NH3/NH4+transporters is more evident in respiratory than in metabolic acidosis. These data show a link among chronic hypercapnia, NH4+excretion and expression of the NH3/NH4+transporters, Rhbg and Rhcg. These studies are important to understand the renal mechanisms of correcting respiratory and metabolic acidosis.Support or Funding InformationU54 GM104940 from NIH, VA Merit grant, NIH‐RO1, Carol Lavin Bernick grant and Tulane Bridge grantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Renal Na+ excretion consequent to pharmacogenetic activation of Gq-DREADD in principal cells.

Stimulation of metabotropic Gq-coupled purinergic P2Y2 receptors decreases activity of the epithelial Na+ channel (ENaC) in renal principal cells of the distal nephron. The physiological consequences of P2Y2 receptor signaling disruption in the P2Y2 receptor knockout mouse are decreased Na+ excretion and increased arterial blood pressure. However, because of the global nature of this knockout model, the quantitative contribution of ENaC and distal nephron compared with that of upstream renal vascular and tubular elements to changes in urinary excretion and arterial blood pressure is obscure. Moreover, it is uncertain whether stimulation of P2Y2 receptor inhibition of ENaC is sufficient to drive renal (urinary) Na+ excretion (UNaV). Here, using a pharmacogenetic approach and selective agonism of the P2Y2 receptor, we test the sufficiency of targeted stimulation of Gq signaling in principal cells of the distal nephron and P2Y2 receptors to increase UNaV. Selective stimulation of the P2Y2 receptor with the ligand MRS2768 decreased ENaC activity in freshly isolated tubules (as assessed by patch-clamp electrophysiology) and increased UNaV (as assessed in metabolic cages). Similarly, selective agonism of hM3Dq-designer receptors exclusively activated by designer drugs (DREADD) restrictively expressed in principal cells of the distal nephron with clozapine- N-oxide decreased ENaC activity and, consequently, increased UNaV. Clozapine- N-oxide, when applied to control littermates, failed to affect ENaC and UNaV. This study represents the first use of pharmacogenetic (DREADD) technology in the renal tubule and demonstrated that selective activation of the P2Y2 receptor and Gq signaling in principal cells is sufficient to promote renal salt excretion.

Temporal changes in urinary excretion of liver-type fatty acid binding protein (L-FABP) in acute kidney injury model of domestic cats: a preliminary study.

Liver-type fatty acid–binding protein (L-FABP) is a biomarker for the early detection of renal diseases in humans. It is secreted along with cytotoxic oxidation products from proximal tubular epithelial cells under conditions of ischemia and/or oxidative stress. This study examined urinary L-FABP excretion under renal ischemia in feline acute kidney injury (AKI) model. L-FABP excretion increased immediately after renal ischemia/reperfusion, despite the absence of obvious structural damage to the kidneys, in the two AKI model cats studied. L-FABP was detected in the renal tubular lumen immediately after renal ischemia/reperfusion in the two cats, but not in a sham surgery cat. These results suggested that high L-FABP excretion is a pathophysiological response associated with antioxidant defense in proximal tubules with renal ischemia and/or oxidative stress in a feline model.

95 - Changes in urinary excretion of oxidative nucleic acid modifications in the week following Copenhagen Marathon 2018, Denmark

Introduction The relationship between oxidative stress and physical activity is very complex. A single bout of exercise seems to induce oxidative stress, whereas regular exercise has been suggested to reduce oxidative stress. Here, we investigated the changes in oxidative RNA and DNA modifications in the week following a single bout of excessive exercise. Methods This observational study was conducted in relation to the Copenhagen Marathon 2018, Denmark. Male, non-professional runners (n=20) were examined before-, immediate after-, four days after-, and seven days after the marathon. Twenty-four-hour (24h) urine excretion of 8-oxo-7,8-dihydro-guanosine(8-oxoGuo) and 8-oxo-7,8-dihydro-2’-deoxyguanosine(8-oxodG) were determined using ultra performance liquid chromatography-tandem mass spectrometry as a measurement of oxidative RNA and DNA modifications, respectively. Results The runners were healthy, non-smokers with cardiorespiratory fitness level above average ((median (inter quartile range(IQR))): Age: 29.6(24.3;37.2) years, body mass index: 24.3(23.8;25.2) kg/m2, VO2max: 52.7(49.5;55.7) ml/min/kg). The baseline level of 8-oxoGuo and 8-oxodG were (median(IQR)): 33.9(29.2; 38.1) and 27.9(21.4; 30.8) nmol/24h, respectively. The level of 8-oxoGuo presented a marginal trend towards increase immediate after the marathon compared to baseline (Δmedian(IQR): 3.9 (-2.4;8.7) nmol/24h, P=0.12), no difference was found in 8-oxodG (-1.8(-4.3;2.6) nmol/24h, P=0.62). On day four, we observed a decrease in both 8-oxoGuo (-2.1(-8.4;0.1) nmol/24h, P=0.02) and 8-oxodG (-2.5(-4.0;0.1) nmol/24h, P Conclusion A single bout of strenuous exercise did not result in immediate increase in oxidative RNA and DNA modifications, however a marginal trend toward increased level of oxidative RNA modifications was observed. Both oxidative RNA and DNA modifications decreased four days after the marathon. The clinical implications of these results remain to be elucidated.

Abstract P224: Urinary Excretion of Sodium, Potassium, Calcium and Magnesium and Blood Pressure Among a Population of ≥ 20-Year-Olds: Evidence From Southwest Coastal Bangladesh

We analyzed data from a stepped wedge randomized trial to evaluate how urinary Na, K, Ca and Mg excretion are associated with the change in blood pressure (BP) among the adult population. We followed up a cohort of 1,191 participants (&gt;20 years old) from 540 households in 16 communities of southwest coastal Bangladesh for five visits during December 2016 - April 2017 when they were exposed to high salinity drinking water. In all visits, we measured participants’ BP (N=5,746) and 24-hour urinary Na, K, Ca and Mg. We used multilevel linear regression models to determine the association among change in urinary excretion of Na, K, Ca and Mg with differences in mean systolic blood pressure (SBP) and diastolic blood pressure (DBP). Models included participant-, household-, and community-level random intercepts and were adjusted for age, sex, BMI, physical activity, smoking status, and household wealth. We restricted analysis among participants who had complete 24-hour urine collection based on measured versus expected urinary creatinine ratio of &gt;=0.7 (N=5,103). The median urinary 24 hours Na excretion was 164 mmol, K was 34 mmol, Ca was 126 mg, and Mg was 83 mg in visit 1. The mean population SBP was 115.6 mmHg in visit 1 and 110.2 in visit 5, and the mean DBP was 68.9 mmHg in visit 1 and 65.9 in visit 5. Compared to visit 1, we found 85% higher urinary Ca in visit 3 and 57% higher urinary Mg in visit 5. We found 100 mmol per 24 hours increase in urinary Na was associated with 1.74 (95% CI: 0.85, 2.62) mmHg higher SBP and 0.51 (95% CI: -0.06, 1.08) mmHg higher DBP, and 50 mmol per 24 hours increase in urinary K was associated with 2.39 (95% CI: 0.84, 3.94) mmHg lower SBP and 0.79 (95% CI: -1.76, 0.17) mmHg lower DBP. We found 100 mg per 24 hours increase in urinary Ca was associated with 0.29 (95% CI: 0.02, 0.60) mmHg lower SBP and 0.32 (0.07, 0.57) mmHg lower DBP, and 100 mg per 24 hours increase in urinary Mg was associated with 1.09 (95% CI: 0.55, 1.64) mmHg lower SBP and 0.41 (0.07, 0.76) mmHg lower DBP. We found SBP lowering effect of urinary Mg increased if urinary Ca was lower (p = 0.048). We found urinary K, Ca, and Mg are associated with lower BP in coastal Bangladesh. Our results suggest a high intake of these beneficial minerals alongside Na reduction may successfully lower mean BP of the population.