Choline supplementation for preterm infants: metabolism of four Deuterium-labeled choline compounds.

BACKGROUND: Supply of choline is not guaranteed in current preterm infant nutrition. Choline serves in parenchyma formation by membrane phosphatidylcholine (PC), plasma transport of poly-unsaturated fatty acids (PUFA) via PC, and methylation processes via betaine. PUFA-PC concentrations are high in brain, liver and lung, and deficiency may result in developmental disorders. We compared different deuterated (D9-) choline components for kinetics of D9-choline, D9-betaine and D9-PC. METHODS: Prospective study (1/2021-12/2021) in 32 enterally fed preterm infants (28 0/7-32 0/7 weeks gestation). Patients were randomized to receive enterally a single dose of 2.7 mg/kg D9-choline-equivalent as D9-choline chloride, D9-phosphoryl-choline, D9-glycerophosphorylcholine (D9-GPC) or D9-1-palmitoyl-2-oleoyl-PC(D9-POPC), followed by blood sampling at 1 + 24 h or 12 + 60 h after administration. Plasma concentrations were analyzed by tandem mass spectrometry. Results are expressed as median (25th/75th percentile). RESULTS: At 1 h, plasma D9-choline was 1.8 (0.9/2.2) µmol/L, 1.3 (0.9/1.5) µmol/L and 1.2 (0.7/1.4) µmol/L for D9-choline chloride, D9-GPC and D9-phosphoryl-choline, respectively. D9-POPC did not result in plasma D9-choline. Plasma D9-betaine was maximal at 12 h, with lowest concentrations after D9-POPC. Maximum plasma D9-PC values at 12 h were the highest after D9-POPC (14.4 (9.1/18.9) µmol/L), compared to the other components (D9-choline chloride: 8.1 [5.6/9.9] µmol/L; D9-GPC: 8.4 (6.2/10.3) µmol/L; D9-phosphoryl-choline: 9.8 (8.6/14.5) µmol/L). Predominance of D9-PC comprising linoleic, rather than oleic acid, indicated fatty-acyl remodeling of administered D9-POPC prior to systemic delivery. CONCLUSION: D9-Choline chloride, D9-GPC and D9-phosphoryl-choline equally increased plasma D9-choline and D9-betaine. D9-POPC shifted metabolism from D9-betaine to D9-PC. Combined supplementation of GPC and (PO) PC may be best suited to optimize choline supply in preterm infants. Due to fatty acid remodeling of (PO) PC during its assimilation, PUFA co-supplementation with (PO) PC may increase PUFA-delivery to critical organs. This study was registered (22.01.2020) at the Deutsches Register Klinischer Studien (DRKS) (German Register for Clinical Studies), DRKS00020502. STUDY REGISTRATION: This study was registered at the Deutsches Register Klinischer Studien (DRKS) (German Register for Clinical Studies), DRKS00020502.

Different choline supplement metabolism in adults using deuterium labelling.

BACKGROUND: Choline deficiency leads to pathologies particularly of the liver, brain and lung. Adequate supply is important for preterm infants and patients with cystic fibrosis. We analysed the assimilation of four different enterally administered deuterium-labelled (D9-) choline supplements in adults. METHODS: Prospective randomised cross-over study (11/2020-1/2022) in six healthy men, receiving four single doses of 2.7 mg/kg D9-choline equivalent each in the form of D9-choline chloride, D9-phosphorylcholine, D9-alpha-glycerophosphocholine (D9-GPC) or D9-1-palmitoyl-2-oleoyl-glycero-3-phosphoryl-choline (D9-POPC), in randomised order 6 weeks apart. Plasma was obtained at baseline (t = - 0.1 h) and at 0.5 h to 7d after intake. Concentrations of D9-choline and its D9-labelled metabolites were analysed by tandem mass spectrometry. Results are shown as median and interquartile range. RESULTS: Maximum D9-choline and D9-betaine concentrations were reached latest after D9-POPC administration versus other components. D9-POPC and D9-phosphorylcholine resulted in lower D9-trimethylamine (D9-TMAO) formation. The AUCs (0-7d) of plasma D9-PC concentration showed highest values after administration of D9-POPC. D9-POPC appeared in plasma after fatty acid remodelling, predominantly as D9-1-palmitoyl-2-linoleyl-PC (D9-PLPC), confirming cleavage to 1-palmitoyl-lyso-D9-PC and re-acylation with linoleic acid as the most prominent alimentary unsaturated fatty acid. CONCLUSION: There was a delayed increase in plasma D9-choline and D9-betaine after D9-POPC administration, with no differences in AUC over time. D9-POPC resulted in a higher AUC of D9-PC and virtually absent D9-TMAO levels. D9-POPC is remodelled according to enterocytic fatty acid availability. D9-POPC seems best suited as choline supplement to increase plasma PC concentrations, with PC as a carrier of choline and targeted fatty acid supply as required by organs. This study was registered at Deutsches Register Klinischer Studien (DRKS) (German Register for Clinical Studies), DRKS00020498, 22.01.2020. STUDY REGISTRATION: This study was registered at Deutsches Register Klinischer Studien (DRKS) (German Register for Clinical Studies), DRKS00020498.

Differential metabolism of choline supplements in adult volunteers.

BACKGROUND: Adequate intake of choline is essential for growth and homeostasis, but its supply does often not meet requirements. Choline deficiency decreases phosphatidylcholine (PC) and betaine synthesis, resulting in organ pathology, especially of liver, lung, and brain. This is of particular clinical importance in preterm infants and cystic fibrosis patients. We compared four different choline supplements for their impact on plasma concentration and kinetics of choline, betaine as a methyl donor and trimethylamine oxide (TMAO) as a marker of bacterial degradation prior to absorption. METHODS: Prospective randomized cross-over study (1/2020-4/2020) in six healthy adult men. Participants received a single dose of 550 mg/d choline equivalent in the form of choline chloride, choline bitartrate, α-glycerophosphocholine (GPC), and egg-PC in randomized sequence at least 1 week apart. Blood was taken from t = - 0.1-6 h after supplement intake. Choline, betaine, TMAO, and total PC concentrations were analyzed by tandem mass spectrometry. Results are shown as medians and interquartile range. RESULTS: There was no difference in the AUC of choline plasma concentrations after intake of the different supplements. Individual plasma kinetics of choline and betaine differed and concentrations peaked latest for PC (at ≈3 h). All supplements similarly increased plasma betaine. All water-soluble supplements rapidly increased TMAO, whereas egg-PC did not. CONCLUSION: All supplements tested rapidly increased choline and betaine levels to a similar extent, with egg-PC showing the latest peak. Assuming that TMAO may have undesirable effects, egg-PC might be best suited for choline supplementation in adults. STUDY REGISTRATION: This study was registered at "Deutsches Register Klinischer Studien" (DRKS) (German Register for Clinical Studies), 17.01.2020, DRKS00020454.

Combined choline and DHA supplementation: a randomized controlled trial.

OBJECTIVE: Choline and docosahexaenoic acid (DHA) are essential nutrients for preterm infant development. They are metabolically linked via phosphatidylcholine (PC), a constitutive plasma membrane lipid and the major transport form of DHA in plasma. Plasma choline and DHA-PC concentrations rapidly decline after preterm birth. To improve preterm infant nutrition, we evaluated combined compared to exclusive choline and DHA supplementation, and standard feeding. DESIGN: Randomized partially blinded single-center trial. SETTING: Neonatal tertiary referral center in Tübingen, Germany. PATIENTS: 24 inborn preterm infants < 32 week postmenstrual age. INTERVENTIONS: Standard nutrition (control) or, additionally, enteral choline (30 mg/kg/day), DHA (60 mg/kg/day), or both for 10 days. Single enteral administration of 3.6 mg/kg [methyl-D9-] choline chloride as a tracer at 7.5 days. MAIN OUTCOME MEASURES: Primary outcome variable was plasma choline following 7 days of supplementation. Deuterated and unlabeled choline metabolites, DHA-PC, and other PC species were secondary outcome variables. RESULTS: Choline supplementation increased plasma choline to near-fetal concentrations [35.4 (32.8-41.7) µmol/L vs. 17.8 (16.1-22.4) µmol/L, p < 0.01] and decreased D9-choline enrichment of PC. Single DHA treatment decreased DHA in PC relative to total lipid [66 (60-68)% vs. 78 (74-80)%; p < 0.01], which was prevented by choline. DHA alone increased DHA-PC only by 35 (26-45)%, but combined treatment by 63 (49-74)% (p < 0.001). D9-choline enrichment showed preferential synthesis of PC containing linoleic acid. PC synthesis via phosphatidylethanolamine methylation resulted in preferential synthesis of DHA-containing D3-PC, which was increased by choline supplementation. CONCLUSIONS: 30 mg/kg/day additional choline supplementation increases plasma choline to near-fetal concentrations, dilutes the D9-choline tracer via increased precursor concentrations and improves DHA homeostasis in preterm infants. TRIAL REGISTRATION: clinicaltrials.gov. Identifier: NCT02509728.

Transport of long-chain polyunsaturated fatty acids in preterm infant plasma is dominated by phosphatidylcholine.

BACKGROUND: Docosahexaenoic (C22:6) and arachidonic (C20:4) acids are long-chain polyunsaturated fatty acids (LC-PUFA) essential to neonatal development, being present in the glycerophospholipids of all organs, particularly the brain. In plasma, LC-PUFAs are mainly present in lipoprotein lipids, which are neutral lipids (triglycerides and cholesterol esters) and glycerophospholipids, like choline containing phosphatidylcholine (PC). PURPOSE: To guide future supplementation strategies of C22:6 and C20:4 in combination with choline, we determined the distribution of C20:4 and C22:6 between PC and neutral lipid. METHODS: Preterm infant plasma (N = 59, postmenstrual age [PMA] 33.9 wk (32.4-36.0)) and cord plasma (N = 34, PMA 34.0 wk (30.86-38.4)) were investigated. PC and neutral lipids were extracted and analyzed using tandem mass spectrometry and gas chromatography, respectively. Data are reported as medians and 25th/75th percentiles. RESULTS: In cord blood, C20:4-PC and C22:6-PC comprised 36.1% (34.2-38.6) and 10.2% (8.8-12.8) of total PC, respectively. In preterm infant plasma, values were only 20.8% (19.2-23.1) and 5.7% (5.2-6.0), respectively (p < 0.001 each). Nevertheless, in preterm infant plasma, 80.6% (77.6-83.0) of C20:4 and 86.0% (83.0-88.9) of C22:6 were found in PC. These values exceeded the proportions of C20:4 and C22:6 in PC of cord plasma [71.3% (67.8-72.9) and 79.2% (75.2-85.4), respectively] (p < 0.0001 each). CONCLUSION: Irrespective of the low proportions of C20:4-PC and C22:6-PC in preterm infant plasma lipids, PC is the major transporter for C20:4 and C22:6. Our data support the hypotheses that choline deficiency may impair end-organ availability of these LC-PUFA in preterm infants. Therefore, supplementation of C20:4 and C22:6 might better be accompanied by choline supplementation.

Randomised crossover study on pulse oximeter readings from different sensors in very preterm infants.

OBJECTIVE: In extremely preterm infants, different target ranges for pulse oximeter saturation (SpO2) may affect mortality and morbidity. Thus, the impact of technical changes potentially affecting measurements should be assessed. We studied SpO2 readings from different sensors for systematic deviations. DESIGN: Single-centre, randomised, triple crossover study. SETTING: Tertiary neonatal intensive care unit. PATIENTS: 24 infants, born at <32 weeks' gestation, with current weight <1500 g and without right-to-left shunt via a patent ductus arteriosus. INTERVENTIONS: Simultaneous readings from three SpO2 sensors (Red Diamond (RD), Photoplethysmography (PPG), Low Noise Cabled Sensors (LNCS)) were logged at 0.5 Hz over 6 hour/infant and compared with LNCS as control using analysis of variance. Sensor position was randomly allocated and rotated every 2 hours. Seven different batches each were used. OUTCOMES: Primary outcome was the difference in SpO2 readings. Secondary outcomes were differences between sensors in the proportion of time within the SpO2-target range (90-95 (100)%). RESULTS: Mean gestational age at birth (±SD) was 274/7 (±23/7) weeks, postnatal age 20 (±20) days. 134 hours of recording were analysed. Mean SpO2 (±SD) was 94.0% (±3.8; LNCS) versus 92.2% (±4.0; RD; p<0.0001) and 94.5% (±3.9; PPG; p<0.0001), respectively. Mean SpO2 difference (95% CI) was -1.8% (-1.9 to -1.8; RD) and 0.5% (0.4 to 0.5; PPG). Proportion of time in target was significantly lower with RD sensors (84.8% vs 91.7%; p=0.0001) and similar with PPG sensors (91.1% vs 91.7%; p=0.63). CONCLUSION: There were systematic differences in SpO2 readings between RD sensors versus LNCS. These findings may impact mortality and morbidity of preterm infants, particularly when aiming for higher SpO2-target ranges (eg, 90-95%). TRIAL REGISTRATION NUMBER: DRKS00027285.

Evidence and Perspectives for Choline Supplementation during Parenteral Nutrition-A Narrative Review.

Choline is an essential nutrient, with high requirements during fetal and postnatal growth. Tissue concentrations of total choline are tightly regulated, requiring an increase in its pool size proportional to growth. Phosphatidylcholine and sphingomyelin, containing a choline headgroup, are constitutive membrane phospholipids, accounting for >85% of total choline, indicating that choline requirements are particularly high during growth. Daily phosphatidylcholine secretion via bile for lipid digestion and very low-density lipoproteins for plasma transport of arachidonic and docosahexaenoic acid to other organs exceed 50% of its hepatic pool. Moreover, phosphatidylcholine is required for converting pro-apoptotic ceramides to sphingomyelin, while choline is the source of betaine as a methyl donor for creatine synthesis, DNA methylation/repair and kidney function. Interrupted choline supply, as during current total parenteral nutrition (TPN), causes a rapid drop in plasma choline concentration and accumulating deficit. The American Society for Parenteral and Enteral Nutrition (A.S.P.E.N.) defined choline as critical to all infants requiring TPN, claiming its inclusion in parenteral feeding regimes. We performed a systematic literature search in Pubmed with the terms "choline" and "parenteral nutrition", resulting in 47 relevant publications. Their results, together with cross-references, are discussed. While studies on parenteral choline administration in neonates and older children are lacking, preclinical and observational studies, as well as small randomized controlled trials in adults, suggest choline deficiency as a major contributor to acute and chronic TPN-associated liver disease, and the safety and efficacy of parenteral choline administration for its prevention. Hence, we call for choline formulations suitable to be added to TPN solutions and clinical trials to study their efficacy, particularly in growing children including preterm infants.

Choline and Betaine Levels in Plasma Mirror Choline Intake in Very Preterm Infants.

Choline is essential for cell membrane formation and methyl transfer reactions, impacting parenchymal and neurological development. It is therefore enriched via placental transfer, and fetal plasma concentrations are high. In spite of the greater needs of very low birth weight infants (VLBWI), choline content of breast milk after preterm delivery is lower (median (p25-75): 158 mg/L (61-360 mg/L) compared to term delivery (258 mg/L (142-343 mg/L)). Even preterm formula or fortified breast milk currently provide insufficient choline to achieve physiological plasma concentrations. This secondary analysis of a randomized controlled trial comparing growth of VLBWI with different levels of enteral protein supply aimed to investigate whether increased enteral choline intake results in increased plasma choline, betaine and phosphatidylcholine concentrations. We measured total choline content of breast milk from 33 mothers of 34 VLBWI. Enteral choline intake from administered breast milk, formula and fortifier was related to the respective plasma choline, betaine and phosphatidylcholine concentrations. Plasma choline and betaine levels in VLBWI correlated directly with enteral choline intake, but administered choline was insufficient to achieve physiological (fetus-like) concentrations. Hence, optimizing maternal choline status, and the choline content of milk and fortifiers, is suggested to increase plasma concentrations of choline, ameliorate the choline deficit and improve growth and long-term development of VLBWI.

Body Composition of Preterm Infants following Rapid Transition to Enteral Feeding.

OBJECTIVE: This study aimed to evaluate body composition at the time of hospital discharge in very preterm infants following rapid transition to full enteral feeding. STUDY DESIGN: We conducted a prospective, observational, cross-sectional study and included 105 preterm infants <32 gestational age or birth weight <1,500 g, born between April 2015 and December 2020, following rapid transition to full enteral feeding (≥140 mL/kg/day). Fat mass/total body mass (BF%) and fat-free mass (FFM) were measured at the time of hospital discharge using air displacement plethysmography. RESULTS: Median and interquartile range (Q1-Q3) of gestational age at birth (GA) was 27.3 (26.1-28.7) weeks and birth weight 845 (687-990) g. Time to reach full enteral feeding was 5 (5-7) days. At 37.6 weeks (36.1-39.0) postmenstrual age (PMA), BF% was 17.0% (14.9-19.8) and FFM 2,161 g (1,966-2,432). BF% was not associated with GA, and not different between small and appropriate for gestational age infants. FFM was significantly lower in infants born small for gestational age. CONCLUSIONS: Following rapid transition to full enteral feeding, FFM and BF% at discharge were similar to other preterm populations. BF% and FFM were not associated with GA at birth but with PMA at measurement. FFM was lower and BF% higher compared to term infants at birth, suggesting diminished parenchymal growth in preterm infants. Continued monitoring of body composition, metabolic health, and neurological development is needed to study long-term effects.

Choline Kinetics in Neonatal Liver, Brain and Lung-Lessons from a Rodent Model for Neonatal Care.

Choline requirements are high in the rapidly growing fetus and preterm infant, mainly serving phosphatidylcholine (PC) synthesis for parenchymal growth and one-carbon metabolism via betaine. However, choline metabolism in critical organs during rapid growth is poorly understood. Therefore, we investigated the kinetics of D9-choline and its metabolites in the liver, plasma, brain and lung in 14 d old rats. Animals were intraperitoneally injected with 50 mg/kg D9-choline chloride and sacrificed after 1.5 h, 6 h and 24 h. Liver, plasma, lungs, cerebrum and cerebellum were analyzed for D9-choline metabolites, using tandem mass spectrometry. In target organs, D9-PC and D9-betaine comprised 15.1 ± 1.3% and 9.9 ± 1.2% of applied D9-choline at 1.5 h. D9-PC peaked at 1.5 h in all organs, and decreased from 1.5-6 h in the liver and lung, but not in the brain. Whereas D9-labeled PC precursors were virtually absent beyond 6 h, D9-PC increased in the brain and lung from 6 h to 24 h (9- and 2.5-fold, respectively) at the expense of the liver, suggesting PC uptake from the liver via plasma rather than local synthesis. Kinetics of D9-PC sub-groups suggested preferential hepatic secretion of linoleoyl-PC and acyl remodeling in target organs. D9-betaine showed rapid turnover and served low-level endogenous (D3-)choline synthesis. In conclusion, in neonatal rats, exogenous choline is rapidly metabolized to PC by all organs. The liver supplies the brain and lung directly with PC, followed by organotypic acyl remodeling. A major fraction of choline is converted to betaine, feeding the one-carbon pool and this must be taken into account when calculating choline requirements.

Calculating Protein Content of Expressed Breast Milk to Optimize Protein Supplementation in Very Low Birth Weight Infants with Minimal Effort-A Secondary Analysis.

Breast milk does not meet the nutritional needs of preterm infants, necessitating fortification. Breast milk is particularly variable in protein content, hence standardized (fixed dosage) supplementation results in inadequate supply. This was a secondary analysis of 589 breast milk protein content measurements of 51 mothers determined by mid-infrared spectroscopy during a clinical trial of higher versus lower protein supplementation in very low birth weight infants. Mothers (and breast milk samples) were divided into a test (41 mothers) and a validation cohort (10 mothers). In the test cohort, the decrease in protein content by day of lactation was modeled resulting in the breast milk-equation (BME)). In the validation cohort, five supplementation strategies to optimize protein supply were compared: standardized supplementation (adding 1.0 g (S1) or 1.42 g protein/100 mL (S2)) was compared with 'adapted' supplementation, considering variation in protein content (protein content according to Gidrewicz and Fenton (A1), to BME (A2) and to BME with adjustments at days 12 and 26 (A3)). S1 and S2 achieved 5% and 24% of adequate protein supply, while the corresponding values for A1-A3 were 89%, 96% and 95%. Adapted protein supplementation based on calculated breast milk protein content is easy, non-invasive, inexpensive and improves protein supply compared to standardized supplementation.

Randomized Longitudinal Study Comparing Three Nasal Respiratory Support Modes to Prevent Intermittent Hypoxia in Very Preterm Infants.

Nasal continuous positive airway pressure (NCPAP) devices using variable (vf-) and continuous (cf-) flow or synchronized nasal intermittent positive pressure ventilation (s-NIPPV) are used to prevent or treat intermittent hypoxia (IH) in preterm infants. Results concerning which is most effective vary. We aimed to investigate the effect of s-NIPPV and vf-NCPAP compared to cf-NCPAP on the rate of IH episodes. Preterm infants with a gestational age of 24.9-29.7 weeks presenting with IH while being treated with cf-NCPAP were monitored for eight hours, then randomized to eight hours of treatment with vf-NCPAP or s-NIPPV. Data from 16 infants were analyzed. Due to an unexpectedly low sample size, the results were only reported descriptively. No relevant changes in the rate of IH events were detected between cf- vs. vf-NCPAP or between cf-NCPAP vs. s-NIPPV. Although limited by its small sample size, s-NIPPV, vf- and cf-NCPAP seemed to be similarly effective in the treatment of IH in these infants.

Choline Content of Term and Preterm Infant Formulae Compared to Expressed Breast Milk-How Do We Justify the Discrepancies?

Choline/phosphatidylcholine concentrations are tightly regulated in all organs and secretions. During rapid organ growth in the third trimester, choline requirement is particularly high. Adequate choline intake is 17-18 mg/kg/day in term infants, whereas ~50-60 mg/kg/day is required to achieve fetal plasma concentrations in preterm infants. Whereas free choline is supplied via the placenta, other choline carriers characterize enteral feeding. We therefore quantified the concentrations and types of choline carriers and choline-related components in various infant formulae and fortifiers compared to breast milk, and calculated the supply at full feeds (150 mL/kg/day) using tandem mass spectrometry. Choline concentration in formula ranged from values below to far above that of breastmilk. Humana 0-VLB (2015: 60.7 mg/150 mL; 2020: 27.3 mg/150 mL), Aptamil-Prematil (2020: 34.7 mg/150 mL), Aptamil-Prematil HA (2020: 37.6 mg/150 mL) for preterm infants with weights < 1800 g, and Humana 0 (2020: 41.6 mg/150 mL) for those > 1800 g, comprised the highest values in formulae studied. Formulae mostly were rich in free choline or phosphatidylcholine rather than glycerophosphocholine and phosphocholine (predominating in human milk). Most formulae (150 mL/kg/day) do not supply the amounts and physiologic components of choline required to achieve fetal plasma choline concentrations. A revision of choline content in formulae and breast milk fortifiers and a clear declaration of the choline components in formulae is required to enable informed choices.