Gerhard Fusch

Target Fortification of Breast Milk with Fat, Protein, and Carbohydrates for Preterm Infants

OBJECTIVES Fortification of breast milk is an accepted practice for feeding very low birth weight infants, however, fixed dosage enhancement does not address variations in native breast milk. This could lead to deficiencies in calories and macronutrients. We therefore established the infrastructure for target fortification in breast milk by measuring and adjusting fat, protein, and carbohydrate content daily. We analyzed nutrient intake, growth, and safety variables. STUDY DESIGN Each 12-hour batch of breast milk was analyzed using near-infrared spectroscopy. Macronutrients were individually added to routine fortification to achieve final contents for fat (4.4 g), protein (3 g), and carbohydrates (8.8 g) (per 100 mL). Fully breast milk fed healthy very low birth weight infants (<32 weeks) were fed the fortified breast milk for at least 3 weeks. Matched pair analysis of 20 infants fed routinely fortified breast milk was performed using birth weight, gestational age, and postnatal age. RESULTS All 650 pooled breast milk samples required at least 1 macronutrient adjusted. On average, 0.3 ± 0.4 g of fat, 0.7 ± 0.2 g of protein, and 1.2 ± 0.2 g of carbohydrate were added. Biochemistry was normal in the 10 target fortified infants (birth weight: 860 ± 309 g, 26.3 ± 1.6 weeks gestational age); weight gain was 19.9 ± 2.7 g/kg/d; and milk intake was 147 ± 5 mL/kg/d (131 ± 16 kcal/kg/d). Osmolality of fortified breast milk was 436 ± 13 mOsmol/kg. Matched pair analysis of infants indicated a higher milk intake (155 ± 5 mL/kg/d) but similar weight gain (19.7 ± 3.3 g/kg/d). No adverse event was observed. The linear relationship between milk intake and weight gain observed in study babies but not seen in matched controls may be related to the variable composition of breast milk. CONCLUSIONS Daily target fortification can be safely implemented in clinical routine and may improve growth.

Prevalence and frequency of circulating t(14;18)-MBR translocation carrying cells in healthy individuals

The t(14;18) translocation is a common genetic aberration that can be seen as an early step in pathogenesis of follicular lymphoma (FL). The significance of low level circulating t(14;18)‐positive cells in healthy individuals as clonal lymphoma precursors or indicators of risk is still unclear. We determined the age dependent prevalence and frequency of BCL2/IgH rearrangements in 715 healthy individuals ranging from newborns to octo‐ and nonagenarians. These results were compared with number of circulating t(14;18)‐positive cells in 108 FL patients at initial presentation. The overall prevalence of BCL2/IgH junctions in this large sample was 46% (327/715). However, there was a striking dependence upon age. Specifically, among individuals up to 10 years old, none had detectable circulating t(14;18)‐positive cells. In the age groups representing 10–50 years old, we found a steady elevation in the prevalence of BCL2/IgH junctions up to a prevalence of 66%. Further increases of the prevalence in individuals older than 50 years were not seen. The mean frequency of BCL2/IgH junctions in healthy individuals ≥40 years (18–26 × 10−6) was significantly higher than in younger subjects (7–9 × 10−6). Four percent (31/715) of individuals carried more than one t(14;18)‐positive cell per 25,000 peripheral blood mononuclear cells (PBMNCs). In comparison, 108 stage III/IV FL patients had a median number of circulating t(14;18)‐positive malignant FL cells of about 9200/1 million PBMNCs (range 7–1,000,000). These findings will further improve the understanding of the relevance of t(14;18)‐positive cells in healthy individuals as a risk marker toward the development into lymphoma precursors.

Mesenchymal Stromal Cells but Not Cardiac Fibroblasts Exert Beneficial Systemic Immunomodulatory Effects in Experimental Myocarditis

Systemic application of mesenchymal stromal cells (MSCs) in inflammatory cardiomyopathy exerts cardiobeneficial effects. The mode of action is unclear since a sufficient and long-acting cardiac homing of MSCs is unlikely. We therefore investigated the regulation of the immune response in coxsackievirus B3 (CVB3)-induced acute myocarditis after intravenous application of MSCs. Wildtype mice were infected with CVB3 and treated with either PBS, human MSCs or human cardiac fibroblasts intravenously 1 day after infection. Seven days after infection, MSCs could be detected in the spleen, heart, pancreas, liver, lung and kidney, whereby the highest presence was observed in the lung. MSCs increased significantly the myocardial expression of HGF and decreased the expression of the proinflammatory cytokines TNFα, IL1β and IL6 as well as the severity of myocarditis and ameliorated the left ventricular dysfunction measured by conductance catheter. MSCs upregulated the production of IFNγ in CD4+ and CD8+ cells, the number of IL10-producing regulatory T cells and the apoptosis rate of T cells in the spleen. An increased number of CD4+CD25+FoxP3 could be found in the spleen as well as in the circulation. In contrast, application of human cardiac fibroblasts had no effect on the severity of myocarditis and the systemic immune response observed after MSCs-administration. In conclusion, modulation of the immune response in extracardiac organs is associated with cardiobeneficial effects in experimental inflammatory cardiomyopathy after systemic application of MSCs.

Rapid measurement of macronutrients in breast milk: How reliable are infrared milk analyzers? *

SUMMARY Background & aims Significant biological variation in macronutrient content of breast milk is an important barrier that needs to be overcome to meet nutritional needs of preterm infants. To analyze macronutrient content, commercial infrared milk analyzers have been proposed as efficient and practical tools in terms of efficiency and practicality. Since milk analyzers were originally developed for the dairy industry, they must be validated using a significant number of human milk samples that represent the broad range of variation in macronutrient content in preterm and term milk. Aim of this study was to validate two milk analyzers for breast milk analysis with reference methods and to determine an effective sample pretreatment. Current evidence for the influence of (i) aliquoting, (ii) storage time and (iii) temperature, and (iv) vessel wall adsorption on stability and availability of macronutrients in frozen breast milk is reviewed. Methods Breast milk samples (n = 1188) were collected from 63 mothers of preterm and term infants. Milk analyzers: (A) Near-infrared milk analyzer (Unity SpectraStar, USA) and (B) Mid-infrared milk analyzer (Miris, Sweden) were compared to reference methods, e.g. ether extraction, elemental analysis, and UPLC-MS/MS for fat, protein, and lactose, respectively. Results For fat analysis, (A) measured precisely but not accurately (y = 0.55x + 1.25, r2 = 0.85), whereas (B) measured precisely and accurately (y = 0.93x + 0.18, r2 = 0.86). For protein analysis, (A) was precise but not accurate (y = 0.55x + 0.54, r2 = 0.67) while (B) was both precise and accurate (y = 0.78x + 0.05, r2 = 0.73). For lactose analysis, both devices (A) and (B) showed two distinct concentration levels and measured therefore neither accurately nor precisely (y = 0.02x + 5.69, r2 = 0.01 and y = −0.09x + 6.62, r2 = 0.02 respectively). Macronutrient levels were unchanged in two independent samples of stored breast milk (−20 °C measured with IR; −80 °C measured with wet chemistry) over a period of 14 months. Conclusions Milk analyzers in the current configuration have the potential to be introduced in clinical routine to measure fat and protein content, but will need major adjustments.

An Integrated Array of Microfluidic Oxygenators as a Neonatal Lung Assist Device: In Vitro Characterization and In Vivo Demonstration

A miniaturized oxygenator device that is perfused like an artificial placenta via the umbilical vessels may have significant potential to save the lives of newborns with respiratory insufficiency. Recently we presented the concept of an integrated modular lung assist device (LAD) that consists of stacked microfluidic single oxygenator units (SOUs) and demonstrated the technical details and operation of SOU prototypes. In this article, we present a LAD prototype that is designed to accommodate the different needs of term and preterm infants by permitting changing of the number of parallel-stacked microfluidic SOUs according to the actual body weight. The SOUs are made of polydimethylsiloxane, arranged in parallel, and connected though 3D-printed polymeric interconnects to form the LAD. The flow characteristics and the gas exchange properties were tested in vitro using human blood. We found that the pressure drop of the LAD increased linearly with flow rate. Gas exchange rates of 2.4-3.8 μL/min/cm(2) (0.3-0.5 mL/kg/min) and 6.4-10.1 μL/min/cm(2) (0.8-1.3 mL/kg/min) for O2 and CO2 , respectively, were achieved. We also investigated protein adsorption to provide preliminary information on the need for application of anticoagulant coating of LAD materials. Albumin adsorption, as measured by gold staining, showed that surface uptake was evenly distributed and occurred at the monolayer level (>0.2 μg/cm(2) ). Finally, we also tested the LAD under in vivo conditions using a newborn piglet model (body weight 1.65-2.0 kg). First, the effect of an arteriovenous bypass via a carotid artery-to-jugular vein shortcut on heart rate and blood pressure was investigated. Heart rate and mean arterial blood pressure remained stable for extracorporeal flow rates of up to 61 mL/kg/min (101 mL/min). Next, the LAD was connected to umbilical vessels (maximum flow rate of 24 mL/min [10.4 mL/kg/min]), and O2 gas exchange was measured under hypoxic conditions (Fi O2  = 0.15) and was found to be 3.0 μL/min/cm(2) . These results are encouraging and support the feasibility of an artificial placental design for an LAD.

Quantification of lactose content in human and cow's milk using UPLC-tandem mass spectrometry.

A sensitive, accurate, and specific quantitative UPLC-MS/MS method was developed for lactose measurement of cows and human milk and validated with cows milk samples certified by an external laboratory. The new method employs only a dilution of raw cows and human milk for simple preparation with no need to remove protein and fat prior to analysis with UPLC-MS/MS. It was operated in negative mode to detect lactose molecules and labeled (13)C(12)-lactose with the highest sensitivity. The principle advantages of the new LC-MS/MS method were: completed lactose determination in 5 min, absolute recovery of 97-107%, lower limit of detection <5 ng/L, and 99% linearity over the concentration range of 0.7-4.4 mg/L for both cows and human milk. The mean lactose concentration of 51 human milk samples was measured as 56.8 ± 5.5 g/L ranging from 43 to 65 g/L. The described method represents validated lactose analysis with high accuracy and precision for a routine lactose determination in raw human milk.

Parallel assessment of nutrition and activity in athletes: Validation against doubly labelled water, 24-h urea excretion, and indirect calorimetry

Abstract The assessment of nutrition and activity in athletes requires accurate and precise methods. The aim of this study was to validate a protocol for parallel assessment of diet and exercise against doubly labelled water, 24-h urea excretion, and respiratory gas exchange. The participants were 14 male triathletes under normal training conditions. Energy intake and doubly labelled water were weakly associated with each other (r = 0.69, standard error of estimate [SEE] = 304 kcal · day−1). Protein intake was strongly correlated with 24-h urea (r = 0.89) but showed considerable individual variation (SEE = 0.34 g · kg−1 · day−1). Total energy expenditure based on recorded activities was highly correlated with doubly labelled water (r = 0.95, SEE = 195 kcal · day−1) but was proportionally biased. During running and cycling, estimated exercise energy expenditure was highly correlated with gas exchange (running: r = 0.89, SEE = 1.6 kcal · min−1; cycling: r = 0.95, SEE = 1.4 kcal · min−1). High exercise energy expenditure was slightly underestimated during running. For nutrition data, variations appear too large for precise measurements in individual athletes, which is a common problem of dietary assessment methods. Despite the high correlations of total energy expenditure and exercise energy expenditure with reference methods, a correction for systematic errors is necessary for the valid estimation of energetic requirements in individual athletes.

Microbiota at Multiple Body Sites during Pregnancy in a Rural Tanzanian Population and Effects of Moringa-Supplemented Probiotic Yogurt

ABSTRACT The nutritional status of pregnant women is vital for healthy outcomes and is a concern for a large proportion of the worlds population. The role of the microbiota in pregnancy and nutrition is a promising new area of study with potential health ramifications. In many African countries, maternal and infant death and morbidity are associated with malnutrition. Here, we assess the influence of probiotic yogurt containing Lactobacillus rhamnosus GR-1, supplemented with Moringa plant as a source of micronutrients, on the health and oral, gut, vaginal, and milk microbiotas of 56 pregnant women in Tanzania. In an open-label study design, 26 subjects received yogurt daily, and 30 were untreated during the last two trimesters and for 1 month after birth. Samples were analyzed using 16S rRNA gene sequencing, and dietary recalls were recorded. Women initially categorized as nourished or undernourished consumed similar calories and macronutrients, which may explain why there was no difference in the microbiota at any body site. Consumption of yogurt increased the relative abundance of Bifidobacterium and decreased Enterobacteriaceae in the newborn feces but had no effect on the mothers microbiota at any body site. The microbiota of the oral cavity and GI tract remained stable over pregnancy, but the vaginal microbiota showed a significant increase in diversity leading up to and after birth. In summary, daily micronutrient-supplemented probiotic yogurt provides a safe, affordable food for pregnant women in rural Tanzania, and the resultant improvement in the gut microbial profile of infants is worthy of further study.

Target Fortification of Breast Milk: How Often Should Milk Analysis Be Done?

Target fortification (TFO) reduces natural macronutrient variation in breast milk (BM). Daily BM analysis for TFO increases neonatal intensive care unit work load by 10–15 min/patient/day and may not be feasible in all nurseries. The variation of macronutrient intake when BM analysis is done for various schedules was studied. In an observational study, we analyzed 21 subsequent samples of native 24-h BM batches, which had been prepared for 10 healthy infants (gestational age 26.1 ± 1.3 weeks, birth weight: 890 ± 210 g). Levels of protein and fat (validated near-infrared milk analyzer), as well as lactose (UPLC-MS/MS) generated the database for modelling TFO to meet recommendations of European Society for Paediatric Gastroenterology Hepatology and Nutrition. Intake of macronutrients and energy were calculated for different schedules of BM measurements for TFO (n = 1/week; n = 2/week; n = 3/week; n = 5/week; n = 7/week) and compared to native and fixed dose fortified BM. Day-to-day variation of macronutrients (protein 20%, carbohydrate 13%, fat 17%, energy 10%) decreased as the frequency of milk analysis increased and was almost zero for protein and carbohydrate with daily measurements. Measurements two/week led to mean macronutrient intake within a range of ±5% of targeted levels. A reduced schedule for macronutrient measurement may increase the practical use of TFO. To what extent the day-to-day variation affects growth while mean intake is stable needs to be studied.

Establishment of micromethods for macronutrient contents analysis in breast milk

Commercially available milk analysers were originally developed for use in the dairy industry, but they are now used to analyse macronutrient content of breast milk in clinical studies and routine care of the premature or very low birthweight (VLBW) infants. Due to the different composition of cow and breast milk, these devices need to be validated against reference methods before they can be used in daily routine. However, current reference methods require a sample volume of 30-100 mL to analyse fat, protein and lactose. It is not feasible to obtain this volume of milk for research purposes, especially from VLBW infants as lactation may be delayed or impaired and the limited volume of breast milk must be provided to the infant. To support validation of milk analysers in both clinical and research settings, the aim of this study is to establish and validate micromethods for precise macronutrient analysis in small volume of breast milk and conduct a feasibility study of the micromethods as a post-validation. Methods include a modified Mojonnier ether extraction (fat), elemental analysis (protein) and ultra-performance liquid chromatography-tandem mass spectrometry (lactose). We were able to downsize volumes required for analysis of fat, protein and lactose to 1 mL, 260 μL and 100 μL; corresponding coefficients of variation are 1.7, 1.8 and 2.3%, respectively. The presented methods allow for reliable and precise analyses of macronutrients in ≤1.5 mL of breast milk and will be used to validate milk analysers.