Objective This study aimed to investigate human fetal exposure to non-nutritive sweeteners (NNS) by analyzing amniotic fluid and umbilical cord blood. Study Design Concentrations of four NNS (acesulfame-potassium [ace-K], saccharin, steviol glucuronide, and sucralose) were measured in amniotic fluid (n = 13) and cord blood samples (n = 15) using liquid chromatography-mass spectrometry. Amniotic fluid samples were obtained for research purposes at the time of term elective cesarean birth or clinically indicated third trimester amnioreduction at Mercy Hospital for Women (Melbourne, Australia). All except four women were in the fasting state. Cord blood samples were obtained from an independent cohort of newborns whose mothers were enrolled in a separate clinical trial at the National Institutes of Health. Results Ten of 13 amniotic fluid samples contained at least one NNS (ace-K, saccharin, steviol glucuronide, and/or sucralose). Maximum amniotic fluid NNS concentrations of ace-K, saccharin, steviol glucuronide, and sucralose were 78.9, 55.9, 93.5, and 30.6 ng/mL, respectively. Ace-K and saccharin were present in 100% and 80% of the cord blood samples, with maximal concentrations of 6.5 and 2.7 ng/mL, respectively. Sucralose was not detected and steviol glucuronide was not measurable in any of the cord blood samples. Conclusion Our results provide evidence of human transplacental transmission of NNS. Based on results predominantly obtained from rodent models, we speculate that NNS exposure may adversely influence the offsprings' metabolic health. Well-designed, prospective clinical trials are necessary to understand the impact of NNS intake during pregnancy on human development and long-term health. Key Points

Objectives Stevia rebaudiana (Bertoni) is a perennial herb native to South America. Its sweetness (∼200–400 times sweeter than sucrose) results from steviol glycosides, particularly stevioside and rebaudioside A. Steviol glycosides are hydrolyzed in the gastrointestinal tract resulting in steviol, which is incompletely absorbed in the colon. In the liver, steviol is converted into its glucuronide derivative and renally excreted. While the use of stevia leaves and crude extracts is still prohibited in the US, steviol glycosides have been ‘generally recognized as safe’ (GRAS) by the Food and Drug Administration (FDA) in 2008. We aimed to determine whether steviol glycosides and glucuronidation products can be found in biosamples collected as early as 2004. Methods In 38 adults, steviol glycosides and glucuronide were analyzed in plasma and in corresponding cerebrospinal fluid samples (CSF); additional 2 persons had only CSF tested. Prenatal exposure was determined in biosamples from 28 individuals (13 amniotic fluid, 15 cord blood). We used ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) utilizing a Thermo Scientific Vanquish UPLC and a Thermo Scientific Altis triple quadruple mass spectrometer with heated electrospray ionization (HESI-II, Thermo Scientific) in negative ion mode (2500 V). Results Seven of 38 adults (18%) had detectable steviol glucuronide concentrations (5 in plasma only, 2 in both plasma and CSF). Maximal concentrations in plasma were 805.4 ng/mL and in CSF 3.3 ng/mL. Two of 13 amniotic fluid samples were positive for steviol glucuronide (max. conc. 93.5 ng/mL) and 1 of 15 cord blood samples contained a trace. In contrast to steviol glucuronide, steviol glycoside could not be measured. Conclusions Steviol glucuronide was found in all types of biosamples (plasma, CSF, amniotic fluid and cord blood), most commonly in plasma (18%). This indicates that exposure to steviol metabolites starts in prenatal life and that these metabolites cross various barriers (e.g., blood-CSF, blood-amniotic fluid). Only samples obtained in and after 2008 were positive for steviol glucuronide, which coincides with the FDA approval. Potential health consequences of exposure to stevia metabolites require further study. Funding Sources N/A.

Sepsis, a life-threatening organ dysfunction, results from dysregulated host responses to infection and still has a high incidence and mortality. Although administration of vasopressors to treat septic shock is standard of care, the benefits are not well established. We evaluated the effect of continuous intravenous norepinephrine infusion in a septic cecal ligation and puncture (CLP) mouse model, evaluating systemic hemodynamics and body temperature post-hoc. CLP surgery significantly decreased mean arterial blood pressure (MAP), heart rate, and body temperature within six hours. Continuous norepinephrine infusion (NE+, n = 12) started at the time of CLP surgery significantly increased MAP at 24 and 30 hours and heart rate at 6, 18, 24, and 30 hours after CLP vs CLP alone (NE-, n = 12). However, addition of norepinephrine did not improve survival rate (NE+ n = 34, NE- n = 31). Early (6 hours or earlier, when the animal became visibly sick) MAP did not predict 7-day mortality. However, heart rates at 3 and at 6 hours after CLP/norepinephrine (NE+) were highly predictive of mortality, as also been found in one clinical study. We conclude that limited hemodynamic support can be provided in a mouse sepsis model. We propose that heart rate can be used to stratify severity of illness in rodent preclinical studies of sepsis therapeutics.

Introduction: In adults, epidemiologic studies consistently show negative health outcomes (e.g. insulin resistance, stroke) related to artificial (or non-nutritive) sweetener (NNS) intake. In children, NNS sweetened beverage consumption is associated with higher total energy and sugar intake. In infants, we documented the immediate appearance of NNS in breast milk after mothers consume diet soda. A positive association between prenatal NNS exposure and higher BMI at 1 year of life has been observed in infants whose mothers routinely consumed NNS during pregnancy. In mice, we recently reported marked changes in intestinal microbiome and hepatic detoxification pathways of pups that had been exposed to NNS via their mothers’ intake during pregnancy and lactation. Thus, we conducted a pilot project to determine whether there is direct evidence for prenatal NNS exposure in humans. In future studies, we will investigate effects on health outcomes. Methods: Concentrations of 3 NNS (acesulfame-potassium (ace-K), sucralose and saccharin) were measured with liquid chromatography-mass spectrometry in cord blood samples (n=15) and amniotic fluid samples (n=13). Aspartame cannot be measured because of its prompt metabolism into aspartic acid and phenylalanine. The cord blood samples were obtained from offspring of women enrolled in a sickle cell clinical trial at the NIH, while the amniotic fluid samples had been obtained for clinical purposes during the 3rd trimester. No dietary information was available other than 2 of 13 women were not in the fasting state when undergoing amniocentesis. Results: In the cord blood samples, ace-K and saccharin were present in 12/15 (80%) samples. None of the samples contained sucralose. In the 13 amniotic fluid samples, 10 (77%) samples contained at least one sweetener. One sample was positive for all 3 sweeteners. Maximum concentrations in cord blood were 6.5 ng/mL for ace-K and 2.7 ng/mL for saccharin, while maximum concentrations in amniotic fluid were 78.9 ng/mL for ace-K, 55.9 ng/mL for saccharin, and 30.6 ng/mL for sucralose (non-fasting sample). Most women were in the fasting state before undergoing amniocentesis or giving birth, thus NNS peak concentrations could not be determined in this pilot study. Discussion and Conclusion: 80% of cord blood samples (babies’ blood) and 77% of amniotic fluid samples (reflecting babies’ direct gastrointestinal/lung exposure) contained ace-K, saccharin and/or sucralose. We speculate that NNS exposure may influence in utero growth and development, e.g. sweet taste preference and metabolic pathways. Prospective studies are necessary to test these hypotheses. Results will determine whether current recommendations (or lack thereof) regarding NNS intake during pregnancy and lactation need to be revised.