Empagliflozin in Heart Failure: Regional Nephron Sodium Handling Effects.

SIGNIFICANCE STATEMENT: The effect of sodium-glucose cotransporter-2 inhibitors (SGLT2i) on regional tubular sodium handling is poorly understood in humans. In this study, empagliflozin substantially decreased lithium reabsorption in the proximal tubule (PT) (a marker of proximal tubular sodium reabsorption), a magnitude out of proportion to that expected with only inhibition of sodium-glucose cotransporter-2. This finding was not driven by an "osmotic diuretic" effect; however, several parameters changed in a manner consistent with inhibition of the sodium-hydrogen exchanger 3. The large changes in proximal tubular handling were acutely buffered by increased reabsorption in both the loop of Henle and the distal nephron, resulting in the observed modest acute natriuresis with these agents. After 14 days of empagliflozin, natriuresis waned due to increased reabsorption in the PT and/or loop of Henle. These findings confirm in humans that SGLT2i have complex and important effects on renal tubular solute handling. BACKGROUND: The effect of SGLT2i on regional tubular sodium handling is poorly understood in humans but may be important for the cardiorenal benefits. METHODS: This study used a previously reported randomized, placebo-controlled crossover study of empagliflozin 10 mg daily in patients with diabetes and heart failure. Sodium handling in the PT, loop of Henle (loop), and distal nephron was assessed at baseline and day 14 using fractional excretion of lithium (FELi), capturing PT/loop sodium reabsorption. Assessments were made with and without antagonism of sodium reabsorption through the loop using bumetanide. RESULTS: Empagliflozin resulted in a large decrease in sodium reabsorption in the PT (increase in FELi=7.5%±10.6%, P = 0.001), with several observations suggesting inhibition of PT sodium hydrogen exchanger 3. In the absence of renal compensation, this would be expected to result in approximately 40 g of sodium excretion/24 hours with normal kidney function. However, rapid tubular compensation occurred with increased sodium reabsorption both in the loop ( P < 0.001) and distal nephron ( P < 0.001). Inhibition of sodium-glucose cotransporter-2 did not attenuate over 14 days of empagliflozin ( P = 0.14). However, there were significant reductions in FELi ( P = 0.009), fractional excretion of sodium ( P = 0.004), and absolute fractional distal sodium reabsorption ( P = 0.036), indicating that chronic adaptation to SGLT2i results primarily from increased reabsorption in the loop and/or PT. CONCLUSIONS: Empagliflozin caused substantial redistribution of intrarenal sodium delivery and reabsorption, providing mechanistic substrate to explain some of the benefits of this class. Importantly, the large increase in sodium exit from the PT was balanced by distal compensation, consistent with SGLT2i excellent safety profile. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: ClinicalTrials.gov ( NCT03027960 ).

Automation of boron chromatographic purification for δ11 B analysis of coral aragonite.

RATIONALE: To detect the small changes in past pH, the boron isotope ratio of coral carbonates, expressed as the δ11 B value, needs to be both precise and accurate (2sd <<1‰). Boron measurements by Multi-Collector Inductively Coupled Plasma Mass Spectrometry (MC-ICPMS) require the boron to be carefully purified before analysis, which is time consuming, and requires specialist training. Here, we use the prepFAST-MC method that enables the automatic extraction of B (up to 25 ng load) from a CaCO3 matrix. METHODS: Samples were purified using the prepFAST-MC automated system with a ~25-µL column of Amberlite IRA743 resin. Boron isotope measurements were performed by MC-ICPMS. The effects of matrix load, speed of sample loading onto the column, and blank contamination were tested to evaluate the effects on the purification process. The optimised protocol was tested on various standards and samples of aragonite corals. RESULTS: The blank contribution for the approach is ~60 pg and is negligible given our sample size (<0.2% sample size). Efficiency of matrix removal is demonstrated with the addition of up to 1.6 mg of dissolved low-B calcium carbonate to NIST SRM 951 with no impact on the accuracy of δ11 B values. The Japanese Geological Survey Porites reference material JCp-1, boric acid standard NIST SRM 951, and seawater, all processed on the prepFAST-MC system, give δ11 B values within error of literature values (δ11 BJCp-1 = 24.31 ± 0.20‰ (2sd, n = 20); δ11 BNIST 951 = -0.02 ± 0.15‰ (2sd, n = 13) and δ11 BSeawater = 39.50 ± 0.06‰ (2sd, n = 2)). Results obtained from the coral Siderastrea siderea purified with the prepFAST-MC system show an average offset from the manual ion-exchange protocols of Δδ11 B = 0.01 ± 0.28‰ (2sd, n = 12). CONCLUSIONS: Our study demonstrates the capacity of the prepFAST-MC method to generate accurate and reproducible δ11 B values for a range of materials, without fractionation, with efficient matrix removal and with negligible blank contribution.

LC-ICP-MS method for the determination of "extractable copper" in serum.

Copper is an essential element for biological functions within humans and animals. There are several known diseases associated with Cu deficiency or overload, such as Menkes disease and Wilson disease, respectively. A common clinical method for determining extractable Cu levels in serum, which is thought to be potentially dangerous if in excess, is to subtract the value of tightly incorporated Cu in ceruloplasmin from total serum Cu. In this work, an automated sample preparation and liquid chromatography (LC) system was combined with inductively coupled plasma-mass spectrometry (ICP-MS) to determine bound Cu and extractable Cu in serum. This LC-ICP-MS method took 250 s for sample preparation and analysis, followed by a column recondition/system reset, thus, a 6 minute sample-to-sample time including sample preparation. The method was validated using serum collected from either control (Atp7b+/-) or Wilson disease rats (Atp7b-/-). The extractable Cu was found to be 4.0 ± 2.3 µM Cu in healthy control rats, but 2.1 ± 0.6 µM Cu in healthy Wilson rats, and 27 ± 16 µM Cu in diseased Wilson rats, respectively. In addition, the extractable Cu/bound Cu ratio was found to be 6.4 ± 3.5%, 38 ± 29%, and 34 ± 22%, respectively. These results suggest that the developed method could be of diagnostic value for Wilson disease, and possibly other copper related diseases.

Premenopausal overweight women do not lose bone during moderate weight loss with adequate or higher calcium intake.

BACKGROUND: Weight loss is associated with bone loss, but this has not been examined in overweight premenopausal women. OBJECTIVE: The aim of this study was to assess whether overweight premenopausal women lose bone with moderate weight loss at recommended or higher than recommended calcium intakes. DESIGN: Overweight premenopausal women [n = 44; x (+/-SD) age: 38 +/- 6.4 y; body mass index (BMI): 27.7 +/- 2.1 kg/m(2)] were randomly assigned to either a normal (1 g/d) or high (1.8 g/d) calcium intake during 6 mo of energy restriction [weight loss (WL) groups] or were recruited for weight maintenance at 1 g Ca/d intake. Regional bone mineral density and content were measured by dual-energy X-ray absorptiometry, and markers of bone turnover were measured before and after weight loss. True fractional calcium absorption (TFCA) was measured at baseline and during caloric restriction by using a dual-stable calcium isotope method. RESULTS: The WL groups lost 7.2 +/- 3.3% of initial body weight. No significant decrease in BMD or rise in bone turnover was observed with weight loss at normal or high calcium intake. The group that consumed high calcium showed a strong relation (r = 0.71) between increased femoral neck bone mineral density and increased serum 25-hydroxyvitamin D. No significant effect of weight loss on TFCA was observed, and the total calcium absorbed was adequate at 238 +/- 81 and 310 +/- 91 mg/d for the normal- and high-calcium WL groups, respectively. CONCLUSION: Overweight premenopausal women do not lose bone during weight loss at the recommended calcium intake, which may be explained by sufficient amounts of absorbed calcium.

Relationships between personal, indoor, and outdoor exposures to trace elements in PM(2.5).

Twenty-four hour average fine particle concentrations of 23 trace elements (TEs) were measured concurrently in (a) ambient air in three urban neighborhoods (Battle Creek-BCK; East St. Paul-ESP; and Phillips-PHI), (b) air inside residences of participants, and (c) personal air near the breathing zone of healthy, non-smoking adults. The outdoor (O), indoor (I), and personal (P) samples were collected in the Minneapolis/St. Paul metropolitan area over three seasons (Spring, Summer, Fall) using either the federal reference (O) or inertial impactor (I,P) inlets to collect PM(2.5). In addition to descriptive statistics, a hierarchical, mixed-effects statistical model was used to estimate the mutually adjusted effects of monitor location, community, and season on mean differences between monitoring locations while accounting for within-subject and within-monitoring period correlation. The relationships among P, I, and O concentrations varied across TEs. The O concentrations were usually higher than P or I for elements like Ca and Al that originate mainly from entrained crustal material, while P concentrations were often highest for other elements with non-crustal sources. Unadjusted mixed model results demonstrated that O monitors more frequently underestimated than overestimated P TE exposures for elements associated with non-crustal sources. This finding was true even though the O TE measurements were taken in the same neighborhoods as the P and I measurements. Further adjustment for community or season effects in the mixed models reduced the number of significant O-P and O-I differences compared to unadjusted models, but still indicated a tendency for underestimation of personal and indoor TE exposures by central site monitors, particularly in the PHI community. These results indicate that community and season are important covariates for developing long term TE exposure estimates, and that personal exposure to trace elements in PM(2.5) is likely to be underestimated by outdoor central site monitors.

Weight loss and calcium intake influence calcium absorption in overweight postmenopausal women.

BACKGROUND: Weight loss (WL) reduces bone mass and increases fracture risk. Mechanisms regulating calcium metabolism during WL are unclear. OBJECTIVE: The objective was to assess the effect of 6 wk of WL at 2 different amounts of calcium intake [normal (NlCa): 1 g/d; high (HiCa): 1.8 g/d] on true fractional calcium absorption (TFCA), bone turnover, and bone-regulating hormones in overweight postmenopausal women. DESIGN: Seventy-three women (body mass index, 26.9 +/- 1.9 kg/m(2)) were recruited either to consume a moderately energy-restricted diet (WL group) or to maintain their body weight [weight-maintenance (WM) group] and were randomly assigned to either the HiCa or the NlCa group in a double-blind manner. Subjects underwent weekly diet counseling, and measurements were taken at baseline and after 6 wk. RESULTS: Fifty-seven women completed the study and had a baseline TFCA of 24.9 +/- 7.4%. Energy restriction significantly decreased the total calcium absorbed (P < 0.05) in the WL group (n = 32) compared with the WM group (n = 25; analysis of covariance). Regression analysis showed that a greater rate of weight loss suppressed TFCA and the total calcium absorbed (P < 0.05) in the HiCa group. The women in the NlCa WL group absorbed inadequate amounts of calcium (195 +/- 49 mg/d), whereas the women in the HiCa WL group absorbed adequate amounts (348 +/- 118 mg/d). Parathyroid hormone explained 22% of the variance in calcium absorbed in the NlCa group only. CONCLUSIONS: We suggest that WL is associated with elevated calcium requirements that, if not met, could activate the calcium-parathyroid hormone axis to absorb more calcium. Normal intakes of calcium during energy restriction result in inadequate total calcium absorption and could ultimately compromise calcium balance and bone mass.

Vitamin D supplementation and calcium absorption during caloric restriction: a randomized double-blind trial.

BACKGROUND: Weight loss (WL) is associated with a decrease in calcium absorption and may be one mechanism that induces bone loss with weight reduction. OBJECTIVE: Because vitamin D supplementation has been shown to increase true fractional calcium absorption (TFCA), the goal of this study was to examine the effect of vitamin D during WL or weight maintenance (WM). DESIGN: A randomized, placebo-controlled, double-blind 6-wk study was conducted in 82 postmenopausal women [BMI (in kg/m(2); ±SD): 30.2 ± 3.7] with 25-hydroxyvitamin D [25(OH)D] concentrations <70 nmol/L during either WL or WM. All women were given 10 µg vitamin D(3)/d and 1.2 g Ca/d and either weekly vitamin D(3) (375 µg) or a placebo equivalent to 63 µg (2500 IU)/d and 10 µg (400 IU)/d, respectively. We measured TFCA with the use of dual-stable isotopes, 25(OH)D, parathyroid hormone, estradiol, calcitriol, and urinary calcium at baseline and 6 wk in weight loss and vitamin D(3)-supplementation (WL-D; n = 19), weight maintenance and vitamin D(3)-supplementation (WM-D; n = 20), weight loss and placebo (n = 22), and weight maintenance and placebo (n = 21) groups. RESULTS: WL groups lost 3.8 ± 1.1% of weight with no difference between vitamin D(3) supplementation and the placebo. The rise in serum 25(OH)D was greatest in the WL-D group (19.8 ± 14.5 nmol/L) compared with in WM-D (9.1 ± 10.3 nmol/L) and placebo groups (1.5 ± 10.9 nmol/L). TFCA increased with vitamin D(3) supplementation compared with placebo treatment (P < 0.01) and decreased during WL compared with WM. Serum 25(OH)D or 1,25-dihyroxyvitamin D did not correlate with TFCA. CONCLUSION: These data show that vitamin D supplementation increases TFCA and that WL decreases TFCA and suggest that, when calcium intake is 1.2 g/d, either 10 or 63 µg vitamin D/d is sufficient to maintain the calcium balance. This trial was registered at clinicaltrials.gov as NCT00473031.

Effect of alendronate and vitamin D3 on fractional calcium absorption in a double-blind, randomized, placebo-controlled trial in postmenopausal osteoporotic women.

Menopause and increasing age are associated with a decrease in calcium absorption that can contribute to the pathogenesis of osteoporosis. We hypothesized that alendronate plus vitamin D(3) (ALN + D) would increase fractional calcium absorption (FCA). In this randomized, double-blind, placebo-controlled multicenter clinical trial, 56 postmenopausal women with 25-hydroxyvitamin D [25(OH)D] concentrations of 25 ng/mL or less and low bone mineral density (BMD) received 5 weekly doses of placebo or alendronate 70 mg plus vitamin D(3) 2800 IU (ALN + D). Calcium intake was stabilized to approximately 1200 mg/d prior to randomization. FCA was determined using a dual-tracer stable-calcium isotope method. FCA and 25(OH)D were similar between treatment groups at baseline (0.31 ± 0.12 ng/mL and 19.8 ± 4.7 ng/mL, respectively). After 1 month of treatment, subjects randomized to ALN + D experienced a significant least squares (LS) mean [95% confidence interval (CI)] increase in FCA [0.070 (0.042, 0.098)], whereas FCA did not change significantly in the placebo group [-0.016 (-0.044, 0.012)]. After ALN + D treatment, patients had higher 25(OH)D levels (LS mean difference 7.3 ng/mL, p < .001). The rise in serum 1,25-dihydroxyvitamin D(3) (p < .02) and parathyroid hormone (p < .001) were greater in the ALN + D group than in placebo-treated patients. ALN + D was associated with an increase in FCA of 0.07. To our knowledge, there is no other trial showing such a marked rise in calcium absorption owing to treatment with a bisphosphonate or owing to a small rise in 25(OH)D. This unique response of ALN + D is important for the treatment of osteoporosis, but the exact mechanism requires further study.

True fractional calcium absorption is decreased after Roux-en-Y gastric bypass surgery.

OBJECTIVE: Roux-en-Y gastric bypass (RYGB) is considered to be the gold standard alternative treatment for severe obesity. Weight loss after RYGB results primarily from decreased food intake. Inadequate calcium (Ca) intake and metabolic bone disease can occur after gastric bypass. To our knowledge, whether malabsorption of Ca contributes to an altered Ca metabolism in the RYGB patient has not been addressed previously. RESEARCH METHODS AND PROCEDURES: We recruited 25 extremely obese women in order to study true fractional Ca absorption (TFCA) before and 6 months after RYGB surgery, using a dual stable isotope method ((42)Ca and (43)Ca) and test load of Ca (200 mg). Hormones regulating Ca absorption and markers of bone turnover were also measured. RESULTS: In 21 women (BMI 52.7 +/- 8.3 kg/m(2), age 43.9 +/- 10.4 years) who successfully completed the study, TFCA decreased from 0.36 +/- 0.08 to 0.24 +/- 0.09 (p < 0.001) after RYGB. Bone turnover markers increased significantly (p < 0.01). TFCA correlated with estradiol levels (r = 0.512, p < 0.02) and tended to correlate with 1,25 (OH)(2)D (r = 0.427, p < 0.06) at final measurement. Stepwise linear regression indicated that estradiol explained 62% of the variance for TFCA at 6 months post-surgery (p < 0.01). DISCUSSION: TFCA decreases (0.12 +/- 0.08) after RYGB surgery but remains within normal range. Although only some patients were estimated to have low Ca absorption after surgery, all of the patients showed a dramatic increase in markers of bone resorption. The alteration in Ca metabolism after RYGB-induced weight loss appears to be regulated primarily by estradiol levels and might ultimately affect bone mass.