Changes In Osmolality Research Articles

Hyperosmotic Stress Induces the Expression of Organic Osmolyte Transporters in Porcine Intestinal Cells and Betaine Exerts a Protective Effect on the Barrier Function.

Background/objectives: The porcine intestinal epithelium plays a fundamental role as a defence interface against pathogens. Its alteration can cause severe inflammatory conditions and diseases. Hyperosmotic stress under physiological conditions and upon pathogen challenge can cause malabsorption. Different cell types counteract the osmolarity increase by accumulating organic osmolytes such as betaine, taurine, and myo-inositol through specific transporters. Betaine is known for protecting cells from hyperosmotic stress and has positive effects when fed to pigs. The aim of this study is to demonstrate the modulation of osmolyte transporters gene expression in IPEC-J2 during osmolarity changes and assess the effects of betaine. Methods: IPEC-J2 were seeded in transwells, where differentiate as a polarized monolayer. Epithelial cell integrity (TEER), oxidative stress (NO) and gene expression of osmolyte transporters, tight junction proteins (TJp) and pro-inflammatory cytokines were evaluated. Results: Cells treated with NaCl hyperosmolar medium (500 mOsm/L) showed a TEER decrease at 3 h and detachment within 24 h, associated with an osmolyte transporters reduction. IPEC-J2 treated with mannitol hyperosmolar medium (500 mOsm/L) upregulated taurine (TauT), myo-inositol (SMIT) and betaine (BGT1) transporters expression. A decrease in TJp expression was associated with a TEER decrease and an increase in TNFα, IL6, and IL8. Betaine could attenuate the hyperosmolarity-induced reduction in TEER and TJp expression, the NO increase and cytokines upregulation. Conclusions: This study demonstrates the expression of osmolyte transporters in IPEC-J2, which was upregulated upon hyperosmotic treatment. Betaine counteracts changes in intracellular osmolarity by contributing to maintaining the epithelial barrier function and reducing the inflammatory condition. Compatible osmolytes may provide beneficial effects in therapies for diseases characterized by inflammation and TJp-related dysfunctions.

7422 Adipsic Diabetes Insipidus Following Pituitary Macroadenoma Resection

Abstract Disclosure: J. Chippior: None. L. Ghalib: None. A 44 year-old female with a PMH of primary hypothyroidism, and a pituitary macroadenoma that was lost to follow up, presented to the ED with a chief complaint of an acute headache and decreased visual acuity involving the right eye over the past year. Patient was found to have a large, homogenous suprasellar mass with the largest dimension measuring 6.3 cm in size, and causing displacement of the optic chiasm. Initial hormonal workup was most notable for elevated prolactin at 128 ng/ml (3.3-26.7 ng/ml), which was slightly lower than when it was previously measured 10 years ago. The patient was taken to the OR for transsphenoidal endoscopic partial resection, but within 24 hours of post-op, she developed hypernatremia at 153 mmol/dL (135-145 mmol/dL) that was suspected to be secondary to diabetes insipidus (DI). In addition, she also suffered several small strokes involving her right midbrain and basal ganglia. She required multiple doses of DDAVP throughout her stay and this was ultimately continued upon discharge. Unfortunately, as follow up MRI had shown residual tumor within the third ventricle, she returned for a 2nd resection but once again suffered additional post-operative complications. The patient experienced additional strokes involving the right frontal and parietal lobes, and her DI became more labile than before, with her serum sodium peaking at 172 mmol/dL. Despite such a high degree of hypernatremia, she did not demonstrate any signs of thirst or any desire to access free water, even after her post-stroke encephalopathy had largely resolved. With less than 100 severe cases documented over the past four decades, it was suspected that the patient had a rare form of DI known as “adipsic DI”. Osmoreceptors within the hypothalamus can trigger the release of ADH or stimulate thirst, with the former requiring a smaller change in serum osmolality to generate such a response. Pituitary surgery involves manipulation of the infundibulum and any inadvertent severing can prevent ADH’s ability to travel from the hypothalamus to the posterior pituitary, subsequently leading to DI. The majority of patients with post-op DI increase their free water intake and subsequently do not develop hypernatremia. Those with adipsic DI lack this compensatory mechanism which leads to the electrolyte disturbance, and carries a higher risk of morbidity and mortality than those with typical DI. Following her second surgery, our patient was discharged with DDAVP once again but this time with a higher dose and frequency. She’s had numerous hospital readmissions since then for altered mental status in the setting of hypernatremia as she still struggles to meet her free water intake and continuously relies on her family for reminders. Though her case is certainly not typical, it highlights the added complexity of managing adipsic DI as it relies not only on DDAVP replacement, but replicating what is supposed to be an innate behavior. Presentation: 6/3/2024

Effect of Human Milk Fortifiers, Medium-chain Triglyceride Oil, Minerals, and Vitamin Supplements on the Osmolality of Pasteurized Donor Human Milk

Aim: We aimed to study the changes in osmolality of pasteurized donor human milk (PDHM) after the addition of four human milk fortifiers (HMFs), medium-chain triglyceride (MCT) oil, minerals, and vitamin supplements commonly used in neonates. Methods: The osmolality of 25 ml of PDHM was measured after the addition of 1 g each of HMF and 0.5 ml of MCT oil separately with a calibrated osmometer. The osmolality of 5 ml of PDHM was measured after the addition of minerals and vitamin supplements in therapeutic doses separately. All these measurements were carried out at 10 min, 30 min, and 2 h by a technician who was blinded to the fortifiers, minerals, and vitamin supplements used. Results: The osmolality of PDHM was 256 mOsm/kg (range = 256–259 mOsm/kg), which increased with all four HMFs tested, but remained below 450 mOsm/kg as recommended by the European Society for Paediatric Gastroenterology Hepatology and Nutrition (ESPGHAN). Proprietary fortifiers increased the osmolality of PDHM to a maximum of 429 mOsm/kg (range = 306–429 mOsm/kg). However, there was no change in the osmolality with MCT oil. A maximum increase in osmolality was observed with the addition of calcium phosphate syrup to PDHM (1181 mOsm/kg). The addition of multivitamins and Vitamin D3 in therapeutic doses increased osmolality (range = 552–753 mOsm/kg) beyond the safety limits of ESPGHAN recommendation. Conclusions: HMFs increased the osmolality of PDHM but were within the safety limits of ESPGHAN recommendation. MCT oil did not affect the osmolality of PDHM. Minerals and vitamin supplements added in therapeutic dosages significantly increased the osmolality of PDHM. Minerals and vitamin supplements need to be appropriately diluted to keep osmolality below 450 mOsm/kg to ensure safety. Future research should focus on manufacturing fortifiers and supplements with low osmolality to ensure safety.

Abstract P144: Estrogen Worsens Kidney Function in Hypertensive Menopausal Rats

Introduction: Before menopause, women generally have lower rates of hypertension and cardiovascular events compared with men. After menopause, women lose significant cardiovascular and renal protection. Estrogen's effect on kidney function is controversial, with some studies showing protection and others, including ours, indicating long-term estrogen exposure causes renal damage. The relationship between estrogen, hypertension, and kidney function is still not fully understood. Aim: Our aim was to investigate the interaction between hypertension and menopause on kidney function and determine whether the timing of disease initiation, pre- or post-menopause, influences these effects. Methods: Female Long-Evans rats were ovariectomized at 46 weeks of age to simulate menopause and treated with estradiol (E2) or vehicle. Some groups were infused with angiotensin II (Ang; 700ng/kg/min), either 4 weeks before or at the time of ovariectomy. Blood pressure (BP), proteinuria, creatinine clearance, osmolality, glomerular (GLOM) area, GLOM collagen deposition, and GPER and ERα receptor by droplet digital PCR were assessed by two-way ANOVA. Results: BP was higher with Ang (P=0.003) but was not affected by E2 (P=0.85) or timing (P=0.49). E2 significantly increased kidney hypertrophy (P<0.001) independent of Ang (P=0.27), especially when hypertension was initiated at the time of menopause (P<0.001). Both E2 and Ang increased water intake and urine output (P<0.05), especially when hypertension was initiated at the time of menopause. Proteinuria, an indicator of renal damage, was significantly higher with E2 (P=0.03), Ang increased GLOM area (P<0.05) with no effect of E2. There was no change in the urinary osmolality due to either Ang or E2. GLOM collagen deposition was not different between groups. In the renal cortex, E2 decreased ERα mRNA (P<0.01), with no difference in GPER. Conclusion: Although E2 did not affect blood pressure, it worsened the impact of hypertension on kidney function, particularly when started at the time of ovariectomy. This may be due to decreased ERα in the cortex and likely unrelated to damage in the glomerulus. Keywords: Menopause, hypertension, renal damage

Plama Osmolality Status in Acute Stroke Patients and in Healthy Individuals in Rajshahi Medical College Hospital

Background: Stroke is the most prevalent neurological emergency and accounts for approximately 50% of all neurological disorders in medical college hospitals. It is the leading cause of adult neurological morbidity and the third leading cause of death globally. Changes in plasma osmolality are associated with increased complications, mortality, morbidity, and disability in stroke patients. Objective: This study aims to investigate the changes in plasma osmolality in different types of acute stroke patients and its impact on stroke outcomes. Method: The study included two groups: Group I comprised 104 acute stroke patients (mean age 58.87±13.70) as cases, and Group II consisted of 104 age- and sex-matched healthy individuals (mean age 57.55±12.60) as controls. Each stroke patient underwent cranial computed tomography within the first 24 hours. Blood tests for blood sugar, serum electrolytes, lipid profile, and blood urea were performed. The CT findings and pathological test results were analyzed using SPSS software. Result: Among the 104 stroke patients, 71 (68.27%) had ischemic stroke, and 33 (31.73%) had hemorrhagic stroke. Dyselectrolytaemia was observed in 73 (70.20%) of the stroke patients, while 31 (29.80%) had normal electrolyte levels. Hyperosmolality was present in 58 (55.77%) of the patients, and hypoosmolality was found in 3 (2.88%). Conclusions: Abnormal plasma osmolality is a significant independent risk factor for complications in acute stroke patients, both ischemic and hemorrhagic. Plasma osmolality estimation should be considered a crucial clinical investigation for evaluating acute stroke patients in the early stages.

Blood, Sweat, and Tears: Implications for Hydration Testing in Combat Sports-Investigating Body Mass Loss and Biomarker Changes.

Combat sports athletes often undergo rapid body mass loss (BML), which presents health risks. Hydration testing has been proposed as a possible solution to reduce or eliminate rapid BML. However, combat sports athletes may exhibit distinct physiological characteristics due to repeated exposure to BML. Thus, traditional and emerging hydration biomarkers should be investigated to determine their potential suitability for field use in this cohort. This study examined whether BML can explain changes in serum and urine osmolality (SosmΔ, UosmΔ), tear osmolarity (TosmΔ), hematocrit (HctΔ), and urine-specific gravity (USGΔ) after mild-moderate passive dehydration. Biomarker reliability was also assessed across two trials. Fifteen male and female combat sports athletes (age: 26.3 ± 5.3 years, body mass: 67.7 ± 9.9 kg) underwent a sauna protocol twice (5-28 days apart) aiming for 4% BML. The average BML in Trials 1 and 2 was 3.0 ± 0.7%. Regression analysis revealed that BML explained HctΔ (R2 = 0.22, p = 0.009) but not SosmΔ (R2 = 0.11, p = 0.079) or other biomarkers. Intraclass correlation coefficients (ICCs) were significant for all biomarkers except TosmΔ (ICC = 0.06, p = 0.37) and post-Tosm (ICC = 0.04, p = 0.42); post-Hct performed best (ICC = 0.82, p < 0.001). Contingency tables with post-Sosm (295 mOsm/kg) and post-USG (1.020) cutoffs revealed an 80% true negative rate (TNR) and a 62% true positive rate (TPR). Increasing the Sosm cutoff to 301 mOsm/kg decreased the TNR to 52% but increased the TPR to 83%. Although blood parameters were most sensitive to BML, they could only explain 11%-22% of biomarker variation. The typical USG cutoff misclassified 42% of athletes postdehydration, and reliability was generally poor-moderate. Alternative strategies should be pursued to manage rapid BML in combat sports.

Effects of Total Parenteral Nutrition on Serum Osmolality and Patent Ductus Arteriosus.

The persistence of high serum osmolality in the early postnatal period is a risk for developing patent ductus arteriosus (PDA). Early aggressive nutrition (EAN), involving total parenteral nutrition (TPN), by which enough concentrations of glucose and amino acids are administered intravenously, is recommended postnatally to improve the neurological prognosis in preterm infants. However, the effects of EAN involving TPN on serum osmolality and the development of a PDA have not been adequately studied. Thus, in this study, we aimed to investigate the impact of TPN on serum osmolality and determine whether increased serum osmolality could be associated with a higher incidence of PDA in preterm infants. In this single-center retrospective observational study, preterm infants born at <28 weeks of gestation who had been admitted to our neonatal intensive care unit (NICU) before (pre-TPN period) and after the introduction of TPN (post-TPN) were included. We reviewed the medical records of these patients, compared the changes in serum osmolality from birth to five days after birth, the clinical background, and the incidence of PDA between these two periods, and analyzed the risk factors. Additionally, the factors affecting the serum osmolality in very preterm infants were examined. The patients who met the intervention criteria of our NICU and received a cyclooxygenase (COX) inhibitor, Indacin® (Nobelpharma, Tokyo, Japan), within seven days after birth were classified as PDA+; those who could not be identified to have PDA flow by echo and did not receive a COX inhibitor were classified as PDA-. The postnatal day and serum sodium (Na+) were statistically significantly correlated with a higher serum osmolality. Serum osmolality remained statistically significantly higher in the PDA+ cohort compared with the PDA- cohort after the first day of life. However, no statistically significant differences were observed in serum osmolality after 24 hours of age, weeks of gestational age, birth weight, or incidence of PDA between the pre- and post-TPN periods. The results of the multiple logistic regression analyses revealed that the increased serum osmolality correlated with PDA development. In this study, the serum Na+ statistically significantly correlated with a higher serum osmolality. Moreover, the increased serum osmolality correlated with PDA development. Thus, the prevention of hypernatremia might reduce the incidence of PDA. Nonetheless, the findings in this study revealed that no statistically significant differences in serum osmolality were observed between the pre-and post-TPN periods, indicating that TPN had little effect on serum osmolality.

Physicochemical Compatibility of Ceftolozane-Tazobactam with Parenteral Nutrition.

Ceftolozane-tazobactam (CT) is used for the treatment of complicated infections and for multidrug-resistant strains of Pseudomonas aeruginosa and extended-spectrum beta-lactamase-producing enterobacteria. In certain cases, simultaneous administration of CT and parenteral nutrition (PN) may be required, but compatibility of Y-site co-administration is unknown. The aim of this study was to analyse the physicochemical compatibility of CT Y-site administered with PN. We evaluated a protocolized PN approach for critical patients in our center. We studied both bolus infusion (2 g ceftolozane/1 g tazobactam in 1 h) and continuous infusion (CI) (6 g ceftolozane/3 g tazobactam) strategies. Samples were visually observed against light, microscopically inspected, and pH was analysed using a pH meter. The mean lipid droplet diameter (MDD) was determined via dynamic light scattering. CT concentration was quantified using HPLC-HRMS. No alterations were observed through visual or microscopic inspection. Changes in pH were ≤0.2, and changes in osmolarity were less than 5%. MDD remained below 500 nm (284.5 ± 2.1 for bolus CT and 286.8 ± 7.5 for CI CT). CT concentrations at t = 0 h and t = 24 h remained within prespecified parameters in both infusion strategies. CT is physiochemically compatible with PN during simulated Y-site administration at the tested concentration and infusion rates.

P-274 Humid or dry culture with medium renewal: impact on A-Grade Day 5 blastocysts. Randomized prospective cohort study

Abstract Study question Could the humid embryo culture impact the development of top-graded blastocysts? Summary answer While no differences were observed in the overall blastocyst development rate, humidity conditions emerged as a significant factor influencing the achievement of A-Grade D + 5 blastocysts. What is known already In recent years, several publications have highlighted the advantages of humid culture (HC) over dry culture (DC) (&amp;lt;45% relative humidity, RH). HC, with its lower risk of media evaporation and consequent osmolality changes. Nevertheless, to the best of our knowledge, this is the first prospective study addressing this aspect through oocytes from the same cohort. Study design, size, duration We have been conducting a randomized control trial (RCT) consisting on 379 MII oocytes from 30 patients from August 2022 until January 2024 (and ongoing). After ICSI procedure, sibling oocytes were divided following 1:1 ratio into both humid (&amp;gt;70% HR) and dry (0% HR) chambers of a Geri incubator (Genea Biomedx, Australia). Ejaculated sperm ICSI cycles with &amp;gt;5 fresh MII were included in the analysis. Time-lapse cleavage timings were manually gathered. Participants/materials, setting, methods Extended culture to the blastocyst stage (D + 5, D + 6 or D + 7) was performed in 80μL of single step G-TL medium (Vitrolife, Sweden). HC remained undisturbed while medium was renewed on D + 3 in DC by changing the culture dish. Fertilization, overall blastocyst development and A-Grade Day 5 blastocysts rates (ASEBIR’s morphology criteria) were compared. Statistical analysis consisted on Chi-squared test, t-student test and logistic regression for computation of Odds Ratio (OR, 95%CI). Main results and the role of chance While no statistically significant differences were observed in the fertilization rate (DC: 76.4%; HC: 69.1%, NS) or the overall usable blastocyst development rate (DC: 48.6%; HC: 53.1%, NS), noteworthy distinctions emerged in the proportion of top-graded embryos based on ASEBIR’s morphology criteria. Specifically, the A-Grade Day 5 blastocyst development rate displayed significant disparities when comparing all fertilized embryos in each culture type (DC: 19.9%; HC: 32.3%; p &amp;lt; 0.05) and also among all usable blastocysts (DC: 40.8%; HC: 60.9%, p &amp;lt; 0,05). The Odds Ratio for achieving A-Grade Day blastocysts depending on type of humidity conditions was 1.87 (1,099 – 3,235) for the humid culture. Moreover, time-lapse imaging facilitated the detection of direct cleavage presence, which was similarly distributed between both incubation types (DC: 27/146; HC: 23/130, NS). However, only 3 embryos showing direct cleavage and cultured in HC reached the blastocyst stage at Day 6, while in dry incubator, 10 embryos with direct cleavage were able to develop to usable blastocysts, three of them being A-Grade on Day 5. Limitations, reasons for caution This study is currently ongoing, aiming to increase the sample size. Future analyses will explore clinical outcomes, especially for underrated embryos, day 6 blastocysts, and those with morphodynamic phenomena. While A-grade blastocyst rate variations may not impact pregnancy outcomes directly, they could influence cumulative success rates. Wider implications of the findings These data highlight that day+3 medium renewal doesn’t equalize conditions to humid culture. Ongoing efforts involve validating these insights through an expanded sample size and extensive outcome analysis. Investigating the long-term impact on clinical outcomes and refining culture protocols can amplify the relevance of these findings in assisted reproductive technologies. Trial registration number pending_protocolOK

No evidence for the melanin desiccation hypothesis in a larval Lepidopteran

Water regulation is an important physiological challenge for insects due to their small body sizes and large surface area to volume ratios. Adaptations for decreasing cuticular water loss, the largest avenue of loss, are especially important. The melanin desiccation hypothesis states that melanin molecules in the cuticle may help prevent water loss, thus offering protection from desiccation. This hypothesis has much empirical support in Drosophila species, but remains mostly untested in other taxa, including Lepidoptera. Because melanin has many other important functions in insects, its potential role in desiccation prevention is not always clear. In this study we investigated the role of melanin in desiccation prevention in the white-lined Sphinx moth, Hyles lineata (Lepidoptera, Sphingidae), which shows high plasticity in the degree of melanin pigmentation during the late larval instars. We took advantage of this plasticity and used density treatments to induce a wide range of cuticular melanization; solitary conditions induced low melanin pigmentation while crowded conditions induced high melanin pigmentation. We tested whether more melanic larvae from the crowded treatment were better protected from desiccation in three relevant responses: i) total water loss over a desiccation period, ii) change in hemolymph osmolality over a desiccation period, and iii) evaporation rate of water through the cuticle. We did not find support for the melanin desiccation hypothesis in this species. Although treatment influenced total water loss, this effect did not occur via degree of melanization. Interestingly, this implies that crowding, which was used to induce high melanin phenotypes, may have other physiological effects that influence water regulation. There were no differences between treatments in cuticular evaporative water loss or change in hemolymph osmolality. However, we conclude that osmolality may not sufficiently reflect water loss in this case. This study emphasizes the context dependency of melanin’s role in desiccation prevention and the importance of considering how it may vary across taxa. In lepidopteran larvae that are constantly feeding phytophagous insects with soft cuticles, melanin may not be necessary for preventing cuticular water loss.

Relative merits of offering a milk replacer, glucose-electrolyte or whey-based diet on the blood composition and health of unweaned calves after transport

The objective of this randomized controlled trial was to assess the relative merits of offering unweaned calves 3 different types of diets to meet energy and water deficits that can occur during journeys. Six young unweaned male Holstein calves were randomly selected from within 2 body weight ranges (median 48 and 42 kg) from each of 29 loads (total n = 174 calves) transported from an auction market or a collection center to a calf sorting center before transport to a veal unit. The calves were then randomly allocated to one of 3 dietary treatments (n = 58 calves/dietary treatment). They were offered either a milk replacer diet (M), a glucose-electrolyte diet (G) or a whey-based diet with added electrolytes (W). The ability of these diets to provide sufficient nutrient energy to restore vigor, avoid hypoglycaemia and clinical signs of dehydration without increasing the risk of diarrhea was assessed. A clinical assessment of dehydration, health and vigor was made, and the calves were blood sampled before feeding, then 2 h and 4 h after feeding. The plasma glucose concentration was increased 2 h and 4 h after feeding the M and W diets. The increases in plasma glucose concentration were greater 2 and 4 h after (a) feeding the M than after the W diet and (b) feeding the M and W diets than after the G diet. Back-transformed means and 95% Confidence Intervals (CI) for the ratio of the plasma glucose concentration at 4 h compared with 0 h for the milk replacer, glucose-electrolytes and whey-based diets were 1.2 mM CI 1.21, 1.35; 0.95 mM CI 0.92, 0.97; and 1.09 mM CI 1.06, 1.14, respectively. There was no effect of diet on the change in serum total protein concentration between before feeding and 2 and 4 h after feeding. The serum osmolality was lower 2 h after feeding the G diet. The fall in serum osmolality was greater 2 h after feeding the G diet than after feeding the M and W diets. The changes in the serum osmolality between before feeding and 2 h after feeding for the milk replacer, glucose-electrolytes and whey-based diets were -0.68 mosmol CI -3.27, 1.91; -5.23 mosmol CI -7.82, and -2.64; -0.13 mosmol CI -2.77, 2.51, respectively. The diet offered at the sorting center had no effect on subsequent growth on the veal rearing farm between arrival and slaughter (milk replacer 1.22 kg/d, CI 1.17, 1.28; glucose-electrolyte diet 1.23 kg/d. CI 1.18, 1.28; whey-based diet 1.28 kg/d CI 1.23, 1.33). The M diet provided the calves with nutrients and water to replace energy and water deficits that had accumulated before arrival at the sorting center, and these dietary benefits were still apparent 4 h after feeding. The benefits of the W diet were similar to those of the M diet, but the M diet was better able to assist the calves in maintaining their plasma glucose concentration 4 h after feeding than the W diet. The G diet had some short-term benefits in providing energy and assistance to the calves to recover from dehydration, as indicated by a decrease in serum osmolality. However, the G diet was clearly inferior to the M and W diets in providing sufficient energy to assist the calves in recovering from the effects of transport and fasting. During the 4-h after feeding, no adverse effects of offering the calves the M or W diets were observed. The benefits of the W diet in replacing energy and water deficits were similar to those of the M diet, but the M diet was better able to assist the calves in maintaining their blood glucose concentration 4 h after feeding than the W diet.

Association of Physicians of India Expert Recommendations on Oral Fluid, Electrolytes, and Energy Management during Transition Care and Discharge for Hospitalized Patients with Nondiarrheal Illnesses.

Acute nondiarrheal illnesses (NDIs) involve overt or subclinical dehydration, requiring rehydration and electrolyte repletion. Dehydration is frequently under-recognized and under-managed, both in outpatient departments (OPDs) and inpatient departments (IPDs). Postadmission dehydration is associated with longer hospital stays and higher inhospital mortality rates. Recognizing and understanding dehydration in hospitalized patients is necessary due to the adverse outcomes associated with this condition. In this article, we aimed to develop practical consensus recommendations on the role of oral fluid, electrolyte, and energy (FEE) management in hospitalized patients with FEE deficits in NDI. The modified Delphi consensus methodology was utilized to reach a consensus. A scientific committee comprising eight experts from India formed the panel. Relevant clinical questions within three major domains were formulated for presentation and discussion: (1) burden and factors contributing to dehydration in hospitalized patients; (2) assessment of fluid and electrolyte losses and increased energy requirements in hospitalized patients; and (3) management of FEE deficits in hospitalized patients [at admission, during intravenous (IV) therapy, IV to oral de-escalation, and discharge]. The consensus level was classified into agreement (mean score ≥4), no consensus (mean score <4), and exclusion (mean score <4 after the third round of discussion). The questions that lacked agreement were discussed during the virtual meeting. The experts agreed that the most common factors contributing to dehydration in patients with NDI hospitalized in IPDs include decreased oral fluid intake, increased fluid loss due to the illness, insensible fluid loss, and a lack of awareness among doctors about dehydration, which can result in poor fluid intake. Time constraints, discontinuity of care, lack of awareness of the principles of fluid balance, lack of formal procedures for enforcing hydration schemes, and lack of adequate training are most often barriers to the assessment of hydration status in hospital settings. Experts used hydration biomarkers, such as changes in body weight, serum, or plasma osmolality; fluid intake; and fluid balance charts; along with urine output, frequency, quantity, and color, to determine hydration status in hospital settings. Experts agreed that appropriate FEE supplementation in the form of ready-to-drink (RTD) fluids can restore FEE deficits and shorten the length of hospital stays in hospitalized patients at admission, during de-escalation from IV to oral therapy, and at discharge. RTD electrolyte solutions with known concentrations of electrolytes and energy are good choices to avoid taste fatigue and replenish FEE in hospitalized patients during transition care and at discharge.

[Translated article] Physicochemical and microbiological stability study of two new preservative-free methylprednisolone eye drops

ObjectiveTo study the physicochemical and microbiological stability over 90 days of two preservative-free methylprednisolone sodium succinate (MTPSS) 1 and 10 mg/mL eye drops for use in ocular pathologies such as Sjögren's syndrome and dry eye syndrome. MethodThe two eye drops were prepared from injectable MTPSS (Solu-moderin® and Urbason®), water for injection and normal saline solution. In accordance with ICH (International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use) guidelines, they were then stored in triplicate under refrigerated conditions (5±3 °C), at room temperature (25±2 °C), and at 40 °C (±2 °C). In accordance with the USP (United States Pharmacopeia), physicochemical controls of the active ingredient content were carried out by HPLC-UV (High Performance Liquid Chromatography with Ultraviolet detection), together with controls of pH, osmolality, and visual examination. Microbiological sterility was also tested under refrigerated conditions up to 30 days in open containers and up to 90 days in closed ones. ResultsThe eye drops stored at 5 °C were the most stable; in the 1 mg/mL eye drops, degradation of the drug fell below 90% from day 21, and in the 10 mg/mL eye drops, from day 42. pH change did not vary by ≥1 unit in formulations stored at 5 °C, unlike the other formulations. Changes in osmolality did not exceed 5% on day 90 in any storage conditions. Samples of non refrigerate eye drops at 10 mg/mL, presented a white precipitate from day 14 and 28, respectively. Non-refrigerated 1 mg/mL eye drops presented suspended particles on day 90. There were no color changes. Microbiological analysis showed that sterility was maintained for over 90 days in the closed containers, although microbial contamination was detected from day 21 in the open containers. Conclusions1 mg/mL MTPSS eye drops show physicochemical and microbiological stability for 21 days under refrigeration, compared to 42 days for 10 mg/mL eye drops stored under the same conditions. However, since they do not include preservatives in their composition, they should not be used for more than 7 days after opening.

A study on the early metabolic effects of salt and fructose consumption: the protective role of water

Increasing serum osmolality has recently been linked with acute stress responses, which over time can lead to increased risk for obesity, hypertension, and other chronic diseases. Salt and fructose are two major stimuli that can induce acute changes in serum osmolality. Here we investigate the early metabolic effects of sodium and fructose consumption and determine whether the effects of sodium or fructose loading can be mitigated by blocking the change in osmolality with hydration. Forty-four healthy subjects without disease and medication were recruited into four groups. After overnight fasting, subjects in Group 1 drank 500 mL of salty soup, while those in Group 2 drank 500 mL of soup without salt for 15 min. Subjects in Group 3 drank 500 mL of 100% apple juice in 5 min, while subjects in Group 4 drank 500 mL of 100% apple juice and 500 mL of water in 5 min. Blood pressure (BP), plasma sodium, and glucose levels were measured every 15 min in the first 2 h. Serum and urine osmolarity, serum uric acid, cortisol, fibroblast growth factor 21 (FGF21), aldosterone, adrenocorticotropic hormone (ACTH) level, and plasma renin activity (PRA) were measured at the baseline and 2 h. Both acute intake of salt or fructose increased serum osmolality (maximum ∼4 mOsm/L peaking at 75 min) associated with a rise in systolic and diastolic BP, PRA, aldosterone, ACTH, cortisol, plasma glucose, uric acid, and FGF21. Salt tended to cause greater activation of the renin-angiotensin-system (RAS), while fructose caused a greater rise in glucose and FGF21. In both cases, hydration could prevent the osmolality and largely block the acute stress response. Acute changes in serum osmolality can induce remarkable activation of the ACTH-cortisol, RAS, glucose metabolism, and uric acid axis that is responsive to hydration. In addition to classic dehydration, salt, and fructose-containing sugars can activate these responses. Staying well hydrated may provide benefits despite exposure to sugar and salt. More studies are needed to investigate whether hydration can block the chronic effects of sugar and salt on disease.

Efficacy of different bioactive glass S53P4 formulations in biofilm eradication and the impact of pH and osmotic pressure

AimThe challenging properties of biofilm-associated infections and the rise of multidrug-resistant bacteria are prompting the exploration of alternative treatment options. This study investigates the efficacy of different bioactive glass (BAG) formulations - alone or combined with vancomycin - to eradicate biofilm. Further, we study the influence of BAG on pH and osmotic pressure as important factors limiting bacterial growth. MethodDifferent BAG S53P4 formulations were used for this study, including (a) powder (<45 μm), (b) granules (500–800 µm), (c) a cone-shaped scaffold and (d) two putty formulations containing granules with no powder (putty A) or with additional powder (putty B) bound together by a synthetic binder. Inert glass beads (1.0–1.3 mm) were included as control. All formulations were tested in a concentration of 1750 mg/ml in Müller-Hinton-Broth against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE). Vancomycin was tested at the minimum-inhibitory concentration for each strain. Changes in pH and osmolality over time were assessed at 0 h, 24 h, 72 h and 168 h. ResultsAll tested BAG formulations showed antibiofilm activity against MRSA and MRSE. Powder and putty B were the most effective formulations suppressing biofilm leading to its complete eradication after up to 168 h of co-incubation, followed by granules, scaffold and putty A. In general, MRSE appeared to be more susceptible to bioactive glass compared to MRSA. The addition of vancomycin had no substantial impact on biofilm eradication. We observed a positive correlation between a higher pH and higher antibiofilm activity. ConclusionsBAG S53P4 has demonstrated efficient biofilm antibiofilm activity against MRSA and MRSE, especially in powder-containing formulations, resulting in complete eradication of biofilm. Our data indicate neither remarkable increase nor decrease in antimicrobial efficacy with addition of vancomycin. Moreover, high pH appears to have a direct antimicrobial impact; the role of high osmolality needs further investigation.

Choroid plexus epithelial cells react to hypertonic KCl by passive cell shrinkage not by NKCC1-mediated water import

Choroid plexus epithelial cells react to changes in osmolarity by passive cell volume changes as other mammalian cells. In previous studies, hyperosmolar challenges resulted in cell shrinkage when either mannitol or NaCl was applied to increase osmolarity. However, a robust cell swelling, despite an outward osmotic gradient, was reported upon challenge with KCl-induced hypertonicity. This was interpreted as inward obligate water transport with Na+, K+, and Cl− mediated by NKCC1. In the present study, single cell volume changes were recorded by pinhole calcein-fluorescence microscopy and contrast imaging before and after 1-minute challenges with osmolarity change. Mannitol- and NaCl-induced hyperosmolarity of 30 mOsm induced the expected ~10% reductions in cell volume by both methods (n=9). Similar KCl-induced hyperosmolarity led to three different responses: cell shrinkage by ~6%, cell swelling by ~50%, or a combined pattern with shrinkage followed by swelling (n=9). Some of the swollen cells started blebbing and/or disrupted, while others partially regained cell volume even after blebbing. KCl-induced cell swelling was sensitive to the NKCC1 inhibitor bumetanide (10 μM), indicating the dependence of the swelling on inward transport by this mechanism. The observation that cell swelling can occur despite a preceding shrinkage indicated that the water influx did not occur against an osmotic gradient. Another observation against NKCC1-mediated water transport was a robust ~14% cell shrinkage induced by 100 mOsm NaCl hyperosmolarity (n=5). Under these conditions, the inward chemical gradient for ions transported by NKCC1 are of similar magnitude as under 15 mOsm KCl-induced hyperosmolarity, where a subset of cells reacted with ~12% swelling (the majority of cells were shrinking, n=5). Thus, the cell swelling is not driven by the combined gradients for Na+, K+, and Cl− into the cells, but is probably secondary to a KCl-induced cell depolarization. Interestingly, cell depolarization by the monovalent cation ionophore gramicidin (10 μM) induces a Na+-dependent cell swelling in a subset of cells with similar magnitude and duration as KCl-induced hyperosmolarity (n=5). Thus, the heterogeneous response to KCl-induced hyperosmolarity may rely on variations in the initial membrane potential among isolated cells. In conclusion, this study can reproduce many of the previous observations regarding cell volume responses to hyperosmolarity in choroid plexus epithelial cells including a bumetanide-sensitive swelling phenomenon in a subset of cells. However, the ionic gradients are not driving this cell swelling, but is rather a consequence of cell depolarization. The research was supported by the Independent Research Fund Denmark | Medical Sciences and Aarhus University Research Foundation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Piezo1 activity is regulated independently in principal and intercalated cells of the renal collecting duct

Water-electrolyte transport in the distal segments of the renal tubule, including the collecting duct (CD), is regulated by mechanical forces arising from changes in fluid flow and osmolarity. Such mechanical stress is known to directly activate Ca2+-permeable Piezo1 channel triggering various downstream effectors under different physiological conditions. However, the functional significance of Piezo1 in morphologically and physiologically distinct principal (PC) and intercalated (IC) cells of the CD is yet to be determined. Here, we implemented electrophysiological and imaging tools to investigate pharmacological Piezo1 activation in PC and IC of freshly isolated intact and split-opened CDs. For this purpose, we applied a variety of systemic treatments, including water restrictions, diuretic injections and dietary manipulations. We observed significantly greater intracellular Ca2+ ([Ca2+]i) signal in PC (AQP2-positive cells) than in IC (AQP2-negative) in freshly isolated split-opened CDs upon application of Piezo1 selective agonists, Yoda-1 and Jedi-2. Patch-clamp analysis in cell-attached configuration allowed us recording of a Yoda-1-activated non-selective channel with 18.6±0.7 pS conductance on both apical and basolateral membranes. These results are in a good agreement with immunofluorescent detection of Piezo1 at both apical and basolateral sides in renal sections and isolated split-opened collecting ducts. Acute stimulation of flow with furosemide injections (2 mg/kgBW, 18h and 2h prior to experimentation) stimulated the elevation of Yoda-1-induced Piezo1-dependent [Ca2+]i elevation in PCs, but not in ICs of split-opened collecting ducts. However, prolonged increased flow in the collecting duct by high K+ diet (for 1 week) decreased Yoda-1-dependent Ca2+ influx in the absence of apparent changes in Piezo1 levels pointing to its inactivation. This led to notable flattening of CD cells, suggesting cytoskeleton reorganization due to increased perfusion pressure existing during high K+ intake. At the same time, anti-diuresis with water restrictions led to reduced Piezo1-dependent [Ca2+]i elevation in PCs of split-opened CDs. Consistently, induction of metabolic acidosis with NH4Cl−supplemented water Piezo1 activity in IC, but not in PC. In summary, our results directly demonstrate functional Piezo1 expression in collecting duct PCs (more) and ICs (less) on both apical and basolateral sides. We also show that acute changes in fluid flow regulate Piezo1 function in PCs, whereas channel activity in ICs responds to systemic acid-base stimuli. This research was supported by NIH-NIDDK DK117865, DK119170, AHA EIA35260097 (to O. Pochynyuk) and AHA-23POST1020372 (to K. Pyrshev). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

ROS formation, mitochondrial potential and osmotic stability of the lamprey red blood cells: effect of adrenergic stimulation and hypoosmotic stress.

Semi-anadromous animals experience salinity fluctuations during their life-span period. Alterations of environmental conditions induce stress response where catecholamines (CA) play a central role. Physiological stress and changes in external and internal osmolarity are frequently associated with increased production of reactive oxygen species (ROS). In this work, we studied the involvement of the cAMP/PKA pathway in mediating catecholamine-dependent effects on osmoregulatory responses, intracellular production of ROS, and mitochondrial membrane potential of the river lamprey (Lampetra fluviatilis, Linnaeus, 1758) red blood cells (RBCs). We also investigated the role of hypoosmotic shock in the process of ROS production and mitochondrial respiration of RBCs. For this, osmotic stability and the dynamics of the regulatory volume decrease (RVD) following hypoosmotic swelling, intracellular ROS levels, and changes in mitochondrial membrane potential were assessed in RBCs treated with epinephrine (Epi, 25 μM) and forskolin (Forsk, 20 μM). Epi and Forsk markedly reduced the osmotic stability of the lamprey RBCs whereas did not affect the dynamics of the RVD response in a hypoosmotic environment. Activation of PKA with Epi and Forsk increased ROS levels and decreased mitochondrial membrane potential of the lamprey RBCs. In contrast, upon hypoosmotic shock enhanced ROS production in RBCs was accompanied by increased mitochondrial membrane potential. Overall, a decrease in RBC osmotic stability and the enhancement of ROS formation induced by β-adrenergic stimulation raises concerns about stress-associated changes in RBC functions in agnathans. Increased ROS production in RBCs under hypoosmotic shock indicates that a decrease in blood osmolarity may be associated with oxidative damage of RBCs during lamprey migration.

Microtubule-associated protein MAP7 promotes tubulin posttranslational modifications and cargo transport to enable osmotic adaptation

Cells remodel their cytoskeletal networks to adapt to their environment. Here, we analyze the mechanisms utilized by the cell to tailor its microtubule landscape in response to changes in osmolarity that alter macromolecular crowding. By integrating live-cell imaging, exvivo enzymatic assays, and invitro reconstitution, we probe the impact of cytoplasmic density on microtubule-associated proteins (MAPs) and tubulin posttranslational modifications (PTMs). We find that human epithelial cells respond to fluctuations in cytoplasmic density by modulating microtubule acetylation, detyrosination, or MAP7 association without differentially affecting polyglutamylation, tyrosination, or MAP4 association. These MAP-PTM combinations alter intracellular cargo transport, enabling the cell to respond to osmotic challenges. We further dissect the molecular mechanisms governing tubulin PTM specification and find that MAP7 promotes acetylation and inhibits detyrosination. Our data identify MAP7 in modulating the tubulin code, resulting in microtubule cytoskeleton remodeling and alteration of intracellular transport as an integrated mechanism of cellular adaptation.

Estudio de estabilidad fisicoquímica y microbiológica de dos nuevos colirios de metilprednisolona sin conservantes

ObjectiveTo study the physicochemical and microbiological stability over 90 days of two preservative-free methylprednisolone sodium succinate (MTPSS) 1 mg/ml and 10 mg/ml eye drops for use in ocular pathologies such as Sjögren’s syndrome and dry eye syndrome. MethodThe two eye drops were prepared from injectable MTPSS (Solu-moderin® and Urbason®), water for injection and normal saline solution. In accordance with ICH (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use) guidelines, they were then stored in triplicate under refrigerated conditions (5 ±3 °C), at room temperature (25 ± 2 °C), and at 40 °C (±2 °C). In accordance with the USP (United States Pharmacopeia), physicochemical controls of the active ingredient content were carried out by HPLC-UV (High Performance Liquid Chromatography with Ultraviolet detection), together with controls of pH, osmolality, and visual examination. Microbiological sterility was also tested under refrigerated conditions up to 30 days in open containers and up to 90 days in closed ones. ResultsThe eye drops stored at 5 °C were the most stable; in the 1 mg/ml eye drops, degradation of the drug fell below 90% from day 21, and in the 10 mg/ml eye drops, from day 42. pH change did not vary by ≥1 unit in formulations stored at 5 °C, unlike the other formulations. Changes in osmolality did not exceed 5% on day 90 in any storage conditions. Samples of non refrigerate eye drops at 10 mg/ml, presented a white precipitate from day 14 and 28 respectively. Non-refrigerated 1 mg/ml eye drops presented suspended particles on day 90. There were no color changes. Microbiological analysis showed that sterility was maintained for over 90 days in the closed containers, although microbial contamination was detected from day 21 in the open containers. Conclusions1 mg/ml MTPSS eye drops show physicochemical and microbiological stability for 21 days under refrigeration, compared to 42 days for 10 mg/ml eye drops stored under the same conditions. However, since they do not include preservatives in their composition, they should not be used for more than 7 days after opening.