Online Resource 1 to:
Choline concentrations are lower in postnatal plasma of preterm infants than in cord
plasma
Wolfgang Bernhard1, MD PhD, Marco Raith1, Rebecca Kunze1, Vera Koch1, Martin Heni2,
MD, Christoph Maas1, MD, Harald Abele, MD3, Christian F. Poets1, MD, Axel R. Franz1,4,
MD
Affiliations: Departments of 1Neonatology, 2Internal Medicine IV and 3Gynecology, and
4
Center for Pediatric Clinical Studies, Faculty of Medicine, Eberhard-Karls-University,
Tübingen, Germany.
Address of correspondance: Wolfgang Bernhard, MD PhD, Department of Neonatology,
Faculty of Medicine, Eberhard-Karls-University, Calwer Straße 7, D-72076 Tuebingen,
Germany; wolfgang.bernhard@med.uni-tuebingen.de; Phone: #-49 7071 29 86377
Short title: Plasma choline concentrations in preterm infants
Methods
Online Resource TABLE 1
Nutrients for enteral and parenteral feeding advancement used in our department during the study
Preterm Human Milk* Preterm Formula** Human Milk Fortifier** Lipid Emulsion*** Fat Soluble Vitamin Emulsion***
per 100ml
per 100ml
per 5g
per 1ml
per 1ml
Protein (g)
1.41
2.3
1
0
0
Fat (g)
3.9
4.2
0.02
0.2
0.1
Carbohydrates (g)
6.6
8.6
0.515
0
0
Choline (mg)
13.7
12.4
0.58
1.1****
1.664****
Folic Acid (mg)
4.3
56
50
0
0
Energy (kcal)
67
80
18
1.9
0.95
Energy (kJ)
280
335
75
8
4
Macro- and Micronutrient content of enteral and parenteral nutrition components during the study period. The following preparations have been
used throughout the study period: preterm formula, BEBA-FN® (Nestlé Nutrition, Frankfurt, Germany); multi component human milk fortifier,
FM85® (Nestlé Deutschland AG, Frankfurt, Germany); fat soluble vitamin emulsion, Vitalipid Infant®, and lipid emulsion, Clinoleic® (both
Baxter Deutschland GmbH, Unterschleißheim, Germany); amino acid solution, Aminoven® 10% (Kabi, Fresenius, Bad Homburg, Germany).
Abbreviations: * = according to [1,2]; ** = enteral supply, data according to manufacturer, *** = parenteral, data according to manufacturer. ****,
Phosphatidylcholine as an emulsifier was the only choline source.
Assessment of sample stability:
The uncertainty whether the values of target analytes of the study, particularly choline, were
(1) artificially affected by sample storage and (2) by differences in stability according to fetal
immaturity was addressed in 19 cord blood samples of preterm and term born infants (9 male,
10 female), with postmenstrual age (PMA) ranging from 26.71-40.29w. Umbilical cord
segments were taken immediately after vaginal delivery or Cesarean section, and 1.5-5mL
blood were drawn into ethylene-diamine-tetraacetate (EDTA) coated blood containers. Aliquots
of 0.20 to 0.25mL were then portioned into 1.5mL polypropylene tubes. One sample was
immediately centrifuged at 2000 x g for 10 min, EDTA plasma supernatant suctioned with a
variable 100µL pipette, and immediately frozen at -20°C. The other samples were either stored
at room temperature (RT) or at 4°C in the refrigerator for up to 24h. At 1, 2, 4 or 24h a sample
aliquot was centrifuged, EDTA plasma aspirated and frozen at -20°C. Samples were then
transferred to a -80°C freezer, and subsequently processed for tandem mass-spectrometric
analysis as described in the main document.
Statistical analysis:
Statistical analysis was performed using Instat®, version 3.10 (Graph Pad, La Jolla, CA,
USA). Data are presented in the form of individual values (Fig. s1) or as medians and
25th/75th percentiles (Fig. s2 and Fig. s3). Regression analysis was performed using runs tests
to control for deviation from linearity, and coefficients of correlation (r) and significance level
(p) indicated.
Result of sample stability testing:
In line with the data in the main document, choline concentrations in cord blood plasma at
time = 0h inversely and linearly correlated with PMA (ONLINE resource Fig. 1) (r = -0.6827;
y-axis intercept=80.82µmol/L (95% confidence interval (53.24-108.40µmol/L) (p=0.0009).
4
The effects of duration of sample storage at 4°C versus room temperature, and of differences
in fetal age at delivery on the changes of choline, betaine and dimethylglycine (DMG)
concentrations are shown in Fig. 2 and Fig. 3 of the ONLINE resource. Here, data are shown
in the form of their deviation during storage for up to 24h, with t=0h set as one. At 4°C
concentrations of choline, betaine or DMG (ONLINE resource Fig. 2A,C and D) did not
significantly change for up to 24h relative to the t=0h value. At room temperature, however,
median choline concentrations rose by 30 (24-40) % within 24h, suggesting that sample
cooling is an essential pre-requisite for sample stability. For up to 4h, these increases were
significant, but only comprised 14 (2-18) % increase (ONLINE resource Fig. 2B). No
significant storage effects were found for betaine and dimethylglycine concentrations at room
temperature (ONLINE resource Fig. 2D and F).
To exclude that our data on higher choline concentrations were due to increased sample
instability in very preterm infants, we correlated the change of choline concentration during
storage with postmenstrual age at delivery. As shown in ONLINE resource Fig. 3A and B,
neither for storage at 4°C nor for room temperature (RT) concentration changes correlated
with PMA (P>0.05). Similarly, there was no significant correlation between PMA and
changes in the concentrations of betaine or DMG (p>0.05, respectively) (data not shown).
Effects of prenatal betamethasone treatment:
As systemic betamethasone treatment is standard to those parturients at risk for preterm
delivery prior to 34 weeks postmenstrual age (PMA), we assessed, whether such treatment
affected the plasma concentrations of choline, betaine and dimethylglycine. Systemic
betamethasone treatment of the parturients prior to delivery did not affect choline
concentrations in cord plasma. However, its betaine concentrations were lower, whereas those
of DMG were higher up to 32w PMA (Fig. r4A-C).
5
References:
1
Holmes-McNary MQ, Cheng WL, Mar MH, Fussell S, Zeisel SH (1996) Choline and
choline esters in human and rat milk and in infant formulas. Am J Clin Nutr 64:572-576.
2
Udipi SA, Kirksey A, West K, Giacoia G (1985) Vitamin B6, vitamin C and folacin
levels in milk from mothers of term and preterm infants during the neonatal period. Am J Clin
Nutr 42:522-530.
6
Online Resource Fig. 1
r = - 0.6827; p=0.0009
Concentration (µmol/L)
60
50
40
30
20
10
0
24
28
32
36
40
44
Postmenstrual Age (weeks)
Fig. r1: Correlation between postmenstrual age and choline concentration in cord
plasma harvested directly after delivery. Data are indicated as medians and 25th /75th
percentiles of n=9 male (open squares) and 10 female (filled squares) individuals of 26.7140.29w postmenstrual age. Data were analyzed with Graph Pad InStat3.0 and linear
regression used after performing runs test to test for departure from linearity.
7
Relative Change (t0=1)
P>0.05
1.0
P>0.05
4°C </=34w
4°C >34w
0.5
2.0
0
4
8 12 16 20 24
C: Betaine, 4°C
1.5
P>0.05
1.0
4°C </=34w
4°C >34w
0.5
0.0
2.0
0
4
P>0.05
8 12 16 20 24
E: DMG, 4°C
1.5
P>0.05
1.0
4°C </=34w
4°C >34w
0.5
0.0
0
4
Relative Change (t0=1)
1.5
0.0
Relative Change (t0=1)
A: Choline, 4°C
Relative Change (t0=1)
2.0
P>0.05
8 12 16 20 24
Time (h)
Relative Change (t0=1)
Relative Change (t0=1)
Online Resource Fig. 2
2.0
B: Choline, RT
1.5
P=0.0098
1.0
P<0.0001
4°C </=34w
4°C >34w
0.5
0.0
2.0
0
4
8 12 16 20 24
D: Betaine, RT
P>0.05
1.5
1.0
P>0.05
4°C </=34w
4°C >34w
0.5
0.0
2.0
0
4
8 12 16 20 24
F: DMG, RT
1.5
P>0.05
1.0
P>0.05
4°C </=34w
4°C >34w
0.5
0.0
0
4
8 12 16 20 24
Time (h)
Fig. r2: Effects of storage time at 4°C (A,C,E) or room temperature (RT) (B,D,F) on
concentration changes of choline (A,B), betaine (C,D) and dimethylglycine (DMG) (E,F)
in cord blood EDTA plasma. Data are indicated as medians and 25th /75th percentiles of n=9
individuals below of 26.7-34.0w PMA (solid line) and n=10 neonates of 34.9-40.3w PMA
(dotted line). Data were analyzed with Graph Pad InStat3.0. Abbreviations: h, hours of EDTA
blood sample storage before centrifugation; P, significance level; w, weeks of postmenstrual
age
8
Online Resource Fig. 3
A: Choline, 4°C
Relative change (t0=1)
2.0
1.5
1.0
1h r = 0.0091, p=0.9744
2h r = 0.1115, p=0.681
0.5
4h
r = -0.1936, p=0.4893
0.0
24
32
36
PMA (w)
40
44
B: Choline, RT
2.0
Relative change (t0=1)
28
1.5
1.0
1h r = 0.1849; p=0.4929;
2h r = 0.1566; p=0.5624
0.5
4h
r = 0.1840; p=0.5116
0.0
24
28
32
36
40
44
PMA (w)
Fig. r3: Relative changes in choline concentration after 1h, 2h or 4h storage at 4°C (A)
or RT (B) relative to postmenstrual age (PMA) at delivery. Data represent changes of
individual values of cord blood EDTA plasma samples (26.7-40.3w PMA) after 1h, 2h or 4h
storage, relative to t=0h. Data were analyzed with Graph Pad InStat3.0 and linear regression
used after performing runs test to test for departure from linearity. Linear regression indicates
no significant effect (p>0.05) of PMA on changes in choline concentrations during this
incubation time at either 4°C (A) or RT (B).
9
Online Resource Fig. 4
Concentration (µmol/L)
150
A: Choline
100
B: Betaine
30
C: Dimethylglycine
80
100
p=0.3763
p<0.0001
60
40
50
20
p=0.0281
10
20
0
–
+
Betamethasone
0
–
+
Betamethasone
0
–
+
Betamethasone
Fig. r4: Effects of prenatal betamethasone treatment on concentrations of choline (A),
betaine (B) and dimethylglycine (C) in cord blood. Data are from preterm infants born
between 24 and 31w+6d gestational age with (N=54) or without (N=36) prenatal systemic
betamethasone treatment. Diamonds indicate individual values, whereas squares and bars
indicate medians and 25th/75th percentiles. Nonparametric correlation (Spearman Rank test),
not assuming Gaussian distribution, was used for analysis. Abbreviation: p, significance level.