ARTICLE
Auteur(s) : Robert J
Pawlosky
Laboratory of Metabolic Control, National Institutes on Alcohol
Abuse & Alcoholism, NIH, Rm 1S-22 5625 Fishers Lane, Bethesda,
MD 20892, Bethesda, Maryland, USA
Introduction
As it has become increasingly accepted by nutritionists that
formulas containing the long chain polyunsaturated fatty acids
(PUFA), 20:4n-6 and 22:6n-3, benefit early development in infants
then guidelines are needed for determining what quantities of these
fatty acids are required in the diet daily to meet the metabolic
demands of newborns [1, 2]. To this end, two compartmental models
were developed using isotope tracer data to assess the
contributions of both 18:2n-6 and 20:3n-6 toward maintaining plasma
concentrations of 20:4n-6 in gestational age-appropriate newborn
infants during the first week of life.
During the early postnatal period placental transfer of
nutrients ceases and infants rapidly develop an increasing capacity
to nurse. Body weight decreases (typically, infants loose 5-10%
body weight during the first week of life) and body fat reserves
are recruited to meet the demand for energy. Consequently, the
balance of energy equilibrium shifts during the early postnatal
period which is likely to have an impact on lipid metabolism until
intake volumes become established.
Two independent compartmental models were developed from the
plasma masses of endogenous n-6 FAs and isotopic tracer data of the
13C-labeled n-6 (from 13C-18:2n-6) and the
2H5-labeled n-6 (from
2H5-20:3n-6) FAs using the WinSAAM (Windows
Simulation, and Analysis Modeling) program. The n-6 FA kinetic
parameters were determined for each subject and mean values were
calculated for the cohort. Quantitative contributions of dietary
18:2n-6 and 20:3n-6 toward maintenance of plasma 20:4n-6 during the
first week of life were determined.
Methods
Subject characteristics and clinical procedures
A description of the subject characteristics and clinical
procedures may be found elsewhere [3] but are briefly outlined
here. Table 1( Table 1 ) gives a brief
description of subject data and feeding regimen. Infants (n = 10),
with gestational ages greater than 34 wk were accepted into the
protocol after receiving informed consent from the mothers and
admitted to the Hospital Sótero del Rio and Clínica Presbiteriana
Madre-Hijo in Santiago, Chile. Feeding was started generally within
2 d after birth, and the type of feeding varied. If breast milk was
unavailable, infants received Similac® infant formula
(Ross Labs, Abbott Park, IL) which contained 18:2n-6 (780 mg
100 mL–1) but devoid of 20:3n-6 and 20:4n-6.
Infants were nursed and/or fed expressed breast milk when
available, and/or Similac® infant formula upon demand.
The quantity of expressed milk and the amount of formula consumed
were determined for each subject. Subjects received a mixture of
13C-U-18:2n-6 (100 mg kg–1) and deuterated
2H5-20:3n-6 (2 mg kg–1).
Blood was drawn (0.5 mL) from an umbilical catheter or from a
peripheral vein, into a tube containing EDTA. Blood was drawn at 0,
4, 8, 24 and 48 h and on the 4th and 7th
d after dosing. Plasma was separated by centrifugation and frozen
at – 80 °C.
Table 1 Subject description and feedind intake data.
|
BW
|
GA
|
|
Age at entrance
|
Wt at entrance
|
Wt at end
|
Age at enteral feeding
|
Formula intake
|
Breast milk intake
|
|
ID
|
(g)
|
(wks)
|
Sex
|
(d)
|
(g)
|
(g)
|
(d)
|
mL/day
|
mL/day
|
|
82
|
3250
|
38
|
F
|
1
|
3400
|
3550
|
4
|
136
|
143
|
|
83
|
3890
|
42
|
M
|
1
|
3930
|
3920
|
2
|
38
|
390
|
|
84
|
3070
|
39
|
M
|
3
|
3080
|
3250
|
3
|
110
|
405
|
|
86
|
2350
|
36
|
M
|
2
|
2390
|
2170
|
4
|
0
|
70
|
|
87
|
3070
|
37
|
M
|
3
|
3060
|
2940
|
3
|
165
|
136
|
|
88
|
3310
|
39
|
M
|
4
|
3510
|
3510
|
4
|
180
|
0
|
|
89
|
3160
|
37
|
M
|
2
|
3410
|
3480
|
3
|
234
|
10
|
|
90
|
2540
|
35
|
M
|
1
|
2550
|
2270
|
2
|
0
|
60
|
|
91
|
4650
|
41
|
F
|
2
|
4580
|
4680
|
3
|
169
|
44
|
|
92
|
2490
|
37
|
M
|
2
|
2440
|
2350
|
3
|
0
|
130
|
Stable isotopes
Carbon-13-uniformly-labeled linoleate (13C-U-18:2n-6,
13C > 95%) and deuterium labeled di-homo-γ-linolenate
(19, 19, 20, 20, 20-2H5-20:3n-6,
2H > 95%) ethyl esters were greater than 95% chemical
purity (Cambridge Isotope Laboratories, Andover, MA).
Lipid extraction and analytical procedures
A complete description of the lipid extraction procedures and gas
chromatography (GC) and GC-mass spectrometry (MS) conditions may be
found elsewhere [4]. Plasma lipids were extracted using a modified
Folch procedure [5]. Plasma lipids were analyzed as their methyl
esters by GC analysis on a polar capillary column with flame
ionization detection. Fatty acids were also derivatized to their
Pentafluorobenzyl esters and analyzed using negative chemical
ionization GC-MS analysis.
Compartmental models
The compartmental models were developed based on the existing
knowledge of fat absorption, n-6 FA metabolism, and circulation of
lipids in blood. Two independent compartmental models of n-6 FA
metabolism (figures
1 and 2)
were developed using the concentration time-courses of the
labeled-FAs and concentrations of endogenous FA in plasma using
WinSAAM (http://www.winsaam.com). The fractional transfer rate
constant coefficient, LI,J, is the fraction of substrate
transferred from substrate-compartment, J, to product-compartment,
I. The units are in h–1. LI,J represents an
assemblage of several independent enzymatic and transport
processes, each having a separate rate constant, for which no
intermediates were isolated. The rate of flow (RI,J)
(table 2( Table 2 )) from
substrate-compartment J to product-compartment I is obtained by
multiplying the mass (MJ) (table 3( Table 3 )) of endogenous FA in compartment J by
LI,J and is given in μg h–1. The percentage
of isotope transferred from J to I is given as PI,J
(table 4( Table 4 )) and is a percent of
the total flux of FA leaving J. PI,J is the fraction of
isotope remaining in the metabolic pathway as opposed to isotope
taken up by tissues or in other ways irreversibly lost from the
compartment.
The compartmental model for 18:2n-6 consisted of six
compartments (figure
1). Compartment 1 represents the dose of the labeled-FA
absorbed through the gastrointestinal tract. Compartments 2, 3, 4,
5 and 7 denote plasma pools of 18:2n-6, 20:3n-6, 20:4n-6, 18:3n-6,
and 20:2n-6. Arrows connecting the six compartments indicate flow
along the path. The rate equations are defined by a set of
differential equations corresponding to flux of labeled-FA through
each respective compartment and those that exit the system.
Table 2 Synthetic and disappearance rates for n-6 fatty
acids in plasma.
|
Disappearances and synthetic rates
|
μg hr–1
|
|
|
82
|
83
|
84
|
86
|
87
|
88
|
89
|
90
|
91
|
92
|
Mean
|
SD
|
cv
|
|
R0,1
|
399340
|
1010500
|
194200
|
268610
|
71826
|
149890
|
274010
|
117800
|
1432300
|
27594
|
394607
|
229863
|
0.58
|
|
R3,2
|
8.8
|
5.2
|
3.8
|
4.0
|
16.4
|
2.1
|
49.2
|
2.9
|
16.0
|
10.4
|
11.9
|
7.0
|
0.59
|
|
R5,2
|
5.0
|
4.1
|
5.7
|
13.8
|
39.4
|
3.3
|
16.0
|
1.7
|
9.0
|
7.1
|
10.5
|
5.6
|
0.53
|
|
R0,2
|
585
|
1427
|
753
|
396
|
219
|
331
|
6027
|
1629
|
6215
|
332
|
1791
|
1165
|
0.65
|
|
R7,2
|
11.4
|
4.8
|
7.8
|
56.6
|
21.2
|
4.6
|
55.3
|
3.0
|
41.7
|
9.3
|
21.6
|
10.7
|
0.50
|
|
R0,7
|
5.8
|
6.3
|
25.9
|
29
|
10.1
|
10.9
|
51.7
|
8.2
|
25.3
|
10.0
|
17.1
|
7.5
|
0.44
|
|
R0,5
|
18.6
|
6.2
|
34.5
|
nd
|
131
|
150
|
58.1
|
257
|
14.9
|
96.1
|
100
|
45
|
0.45
|
|
R4,3
|
25.3
|
20.0
|
23.0
|
2.3
|
33.7
|
11.1
|
87.5
|
56.9
|
76.2
|
56.0
|
39.2
|
14.2
|
0.36
|
|
R0,3
|
0.5
|
6.3
|
59.7
|
143
|
19.0
|
15.4
|
8.7
|
1.0
|
2.5
|
49.0
|
|
22
|
0.73
|
|
R0,4
|
99.3
|
212
|
210
|
2120
|
507
|
68.4
|
662
|
0
|
146
|
211
|
|
315
|
0.74
|
Table 3 Total plasma fatty acids.
|
Plasma fatty acids (μg)
|
|
compartment/n-6 fatty acid
|
Subject ID
|
|
82
|
83
|
84
|
86
|
87
|
88
|
89
|
90
|
91
|
92
|
Mean
|
SD
|
|
M2/18:2
|
16136
|
29212
|
19902
|
11381
|
17700
|
9768
|
20493
|
10025
|
29439
|
28913
|
19287
|
3899
|
|
M3/2O:3
|
3536
|
3424
|
2733
|
3666
|
1891
|
2057
|
3205
|
2393
|
5243
|
5369
|
3359
|
599
|
|
M4/2O:4
|
16829
|
16664
|
9122
|
14285
|
14283
|
8367
|
17419
|
10957
|
29521
|
24377
|
16182
|
3303
|
|
M7/20:2
|
548
|
393
|
752
|
nd
|
747
|
1625
|
645
|
936
|
590
|
1617
|
873
|
325
|
|
M5/18:3
|
485
|
309
|
212
|
288
|
241
|
399
|
517
|
141
|
757
|
310
|
366
|
90
|
Table 4 Percent of labeled fatty acids transferred
through compartments.
|
value *100%
|
|
% flux
|
Subject ID
|
|
82
|
83
|
84
|
86
|
87
|
88
|
89
|
90
|
91
|
92
|
Mean
|
SD
|
cv
|
|
P2,1
|
0.002
|
0.001
|
0.004
|
0.002
|
0.002
|
0.002
|
0.022
|
0.014
|
0.004
|
0.001
|
0.005
|
0.002
|
0.38
|
|
18:2n-6
|
|
P3,2
|
0.008
|
0.003
|
0.007
|
0.029
|
0.133
|
0.011
|
0.003
|
0.010
|
0.001
|
0.177
|
0.038
|
0.033
|
0.85
|
|
LNA ->20:3n-6
|
|
P7,2
|
0.014
|
0.004
|
0.005
|
0.009
|
0.055
|
0.007
|
0.008
|
0.002
|
0.003
|
0.018
|
0.012
|
0,008
|
0.68
|
|
LNA ->20:2n-6
|
|
P5,2
|
0.019
|
0.003
|
0.010
|
0.120
|
0.072
|
0.015
|
0.009
|
0.002
|
0.007
|
0.016
|
0.027
|
0.020
|
0.74
|
|
LNA ->1B:3n-6
|
|
P4,3
|
0.943
|
0.761
|
0.278
|
0.016
|
0.824
|
0.400
|
0.910
|
1.010
|
0.968
|
1.017
|
0.713
|
0.184
|
.26
|
|
20:3n-6 ->20:4n-6
|
Constraints and limits
Plasma n-6 FA concentrations, determined from mean values over
168 h for each subject, were used to represent the mass of
endogenous substrates (MJ) available for biosynthesis
(table 3) and these values were held constant. For purposes of
estimating a daily n-6 FA intake for each subject, the FA content
of the infant formula, availability of breast milk, and frequency
of feeding were entered into the model (table 1). To determine
differences between the efficacy of the two precursors (18:2n-6 and
20:3n-6) toward synthesis of 20:4n-6, a paired t-test analysis was
performed on values of the rate parameters using each subject as
its own control. A p-value of .05 or lower was considered
significant.
Calculations, errors, and predicting dietary n-6 FA intake
Initial LI,J and PI,J estimates, derived from
the concentration-time curves, were adjusted to compensate for
individual variances in plasma data until the model prediction gave
the best fit to the experimental data. Final values were determined
using an iterative non-linear least squares routine. The error
model included assumptions of independence, constant variance, and
normal distribution about zero. Consistent with the precision of
analytical methods, data points were weighted by assigning a
fractional standard deviation of 0.1 to each measurement. Daily
dietary n-6 FA intake values (UJ) (table 5( Table 5 )) were estimated for each infant while
constraining plasma FA masses to known limits. Additionally, the
model was adjusted to compensate for low intake volumes during the
first 48 h after birth with a gradual increase in volume.
Table 5 Predicted daily fatty acid intake amounts.
|
μg hr–1
|
|
Compartment/Fatty acid
|
82
|
83
|
84
|
86
|
87
|
88
|
89
|
90
|
91
|
92
|
Mean
|
SD
|
cv
|
|
U2/18:2
|
285686
|
722857
|
139271
|
192214
|
51394
|
107307
|
200129
|
85336
|
1027571
|
151129
|
296289
|
160108
|
0.57
|
|
U3/20:3
|
6
|
11
|
54
|
98
|
12
|
16
|
34
|
32
|
33
|
68
|
36
|
15
|
0.40
|
|
U4/20:3
|
53
|
137
|
133
|
1513
|
338
|
41
|
410
|
311
|
50
|
111
|
310
|
221
|
0.91
|
|
U5/20:2
|
6
|
1
|
16
|
145
|
52
|
102
|
19
|
159
|
3
|
65
|
57
|
30
|
0.83
|
|
U7/18:3
|
0
|
1
|
13
|
17
|
2
|
5
|
1
|
4
|
1
|
5
|
5
|
3
|
0.57
|
Results and discussion
Ten (8 male and 2 female) infants completed the protocol. Most
received supplemental feeding with breast milk and/or infant
formula in increasing volume during the study. Two 18:2n-6
compartments, one for the isotope administration (GI) and the
second for the appearance of the FA in the plasma were incorporated
into the model (figure
1). Approximately 94% of labeled-18:2n-6 ethyl ester was
absorbed (range: 89-99%) based on the amount of isotope recovered
from the feces over 48 h. Using the area under the curve
calculation (AUC), the mean value of 13C-U-18:2n-6 (±
SD) appearing in the plasma was 254.5 ± 58.5 nmol·mL–1
h. The mean AUC value for 2H5-20:3n-6 (AUC ±
SD) appearing in the plasma was 8.5 ± 3.9 nmol·mL–1 h.
The synthetic and utilization rates, RX,J, (table 2)
represent the total mass of each n-6 FA that exit the substrate
compartment J and is either transferred to product compartment I or
leaves the pathway (0) (but not necessarily the system). The mean
value for turnover of 18:2n-6 through the system was 4.2 g
kg–1 d–1 (CV: 0.58) and the mean turnover of
18:2n-6 in the plasma (R0,2) was 43 mg d–1
(CV 0.65) for the group. The high turnover rate may be associated
with the very early postnatal period and as the intake of breast
milk and/or formula increases this value may moderate reflecting
the change in the lipid composition of the diet [6]. However,
consistent with the present findings a high fractional turnover of
18:2n-6 (mean value 93.7% d–1) was also observed in
adult male subjects [7]. The mean daily turnover in mg
d–1 of the other n-6 FA in the plasma were: 0.41 (CV
0.50), 2.4 (CV 0.49), 0.73 (CV 0.81) and 10.2 (CV 0.74) for
18:3n-6, 20:2n-6, 20:3n-6 and 20:4n-6, respectively. The mean rate
of synthesis of 20:4n-6 from 20:3n-6 (R4,3) was 39.2 μg
h–1 or 0.94 mg d–1 (CV 0.36) from the
13C-FA and 53 μg h–1 (CV 0.48) from the
2H-FA.
The proportion of the plasma n-6 FA PI,J directed
towards biosynthesis was determined and these values are given in
table 4. On average about 0.5% of the administered dose of
13C-18:2n-6 and 0.3% of
2H5-20:3n-6 appeared in the plasma
(P2,1). The total mean percentage of plasma 18:2n-6
directed toward synthesis of all other n-6 FA was approximately
10.3% (range: 1.7-29%, CV 0.62). The mean percentage of plasma
13C-20:3n-6 destined for synthesis of
13C-20:4n-6 was 71% (CV 0.26) (table 4). This contrasts
with a much smaller value (26%, p < .02) of
2H5-20:3n-6 destined for the synthesis of
2H5-20:4n-6 (CV 0.56) (data not shown). This
suggests that that the preferred substrate for 20:4n-6 biosynthesis
is 20:3n-6 arising from 18:2n-6. However, when taking into
consideration the percentage of each labeled substrate appearing in
the plasma, and the overall percent conversion of each precursor to
20:4n-6, then dietary 20:3n-6, as measured by
2H5-20:3n-6 affords approximately a 6-fold
greater delivery of 20:4n-6 compared to 18:2n-6 as measured by
13C-18:2n-6. Sauerwald et al., estimated that the
fractional rate of conversion (FRC) of 18:2n-6 to 20:4n-6 (FRC is
identical to the P-value used here) was between 0.4-1.1% in 3
wk-old infants and these values depended on the α-linolenic acid
content of the formula [8]. In the present study, the net mean FRC
for conversion of 18:2n-6 to 20:4n-6 was 2.7% in these
newborns.
Using the appropriate feeding regimen for each subject (table
1), intake values for 18:2n-6, 20:2n-6, 18:3n-6, 20:3n-6 and
20:4n-6 were calculated (table 5) that were consistent with each
FA’s synthetic and disappearance rates and total plasma
concentration (table 2). The daily mean (± SD) intake of
18:2n-6 and 20:4n-6 were calculated to be 3.0 (± 1.8) g
kg–1 d–1 and 2.8 (± 2.4) mg
kg–1d–1, respectively.
The compartmental model for 18:2n-6 predicted an 18:2n-6 intake
amount of 3.0 g kg–1 d–1 (CV 0.42) with a
turnover rate through the system of 4.2 g kg–1
d–1 (CV 0.58) for these subjects which is consistent
with the plasma concentration of 18:2n-6. This is significant since
results arising from this study form a basis on which to determine
the effects of feeding a particular infant formulation on
maintenance of plasma fatty acid homeostasis. The study also has
the unique capability of isolating and comparing values of
intermediate steps, such as in the conversion of 20:3n-6 to
20:4n-6. However, certain precautions should be considered before
the current compartmental model can be successfully adapted for the
determination of dietary requirements of 18:2n-6 in infants. The
high rate of turnover of 18:2n-6 observed here may only be relevant
to the very early postnatal period reflecting a high demand for
18:2n-6 as an energy resource. Since, during the first few days
after birth intake volumes were low, then it is likely that body
lipid stores supplied the remainder of the 18:2n-6 as the steady
state plasma concentrations did not decrease. As infants become
adjusted to nursing with increased availability of energy-rich
lipids (including medium chain triglycerides) this value may
decrease. The values determined for the percent conversion of
18:2n-6 to 20:4n-6 in the current model were of a similar magnitude
to those observed in 3 wk old infants. Yet these rates of 20:4n-6
synthesis are incapable of sustaining plasma 20:4n-6 concentrations
and an intake of approximately 4 mg kg–1
d–1 is needed to meet this demand, an amount that is
only a fraction of that which is available from human milk.
Acknowledgements
The author acknowledges several collaborators involved in this
study. Ricardo Uauy, Institute of Nutrition and Food Technology
(INTA), Santiago, Chile, and Norman Salem Jr., Laboratory of
Membrane Biochemistry and Biophysics, NIAAA, NIH were co-principal
investigators and responsible for the study design. Adolfo Llanos
and Patricia Mena were responsible for clinical exclusions, subject
care and in specimen collection, Neonatology Unit, Hospital Sótero
del Rio, Santiago, Chile. Yuhong Lin, Laboratory of Membrane
Biochemistry and Biophysics, NIAAA, NIH had overall responsibility
for quality control and preparation of isotopic labeled materials,
analyses of plasma fatty acids and management of the data base.
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development. Acta Paediatr 2001; 90(4): 460-4.
3 Pawlosky RJ, Lin YH, Llanos A, Mena P,
Uauy R, Salem Jr. N. Compartmental analyses of
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