Full text of DOI:10.1007/s11745-001-0757-x

Influence of Formulas with Borage Oil or Borage Oil Plus Fish Oil
on the Arachidonic Acid Status in Premature Infants
Hans Demmelmaira, Franziska Feldla, Imre Horváthb, Tamás Niederlandb, Viktória Ruszinkób,
Daniel Raederstorffc, Cristina De Minc, Reto Mugglic, and Berthold Koletzkoa,*
aKinderklinik and Kinderpoliklinik, Dr. von Haunersches Kinderspital, Ludwig-Maximilians University, D-80336 Munich,
Germany, bPetz Aladár County Teaching Hospital, Department of Pediatrics, H-9002 Gyor, Hungary,
and cF. Hoffmann-La Roche Ltd., Division of Vitamins, CH-4070 Basel, Switzerland
ABSTRACT: Several studies have reported that feeding (1,2). Evidence is accumulating that the availability of appre-
γ-linolenic acid (GLA) has resulted in no increase in arachidonic
acid (AA) in newborns. This result was ascribed to the eico-
sapentaenoic acid (EPA)-rich fish oil used in these formulas.
Docosahexaenoic acid (DHA) sources with only minor amounts
of EPA are now available, thus the addition of GLA to infant for-
mulas might be considered an alternative to AA supplementa-
tion. Sixty-six premature infants were randomized to feeding one
of four formulas [ST: no GLA, no long-chain polyunsaturated
fatty acids; BO: 0.6% GLA (borage oil); BO + FOLOW: 0.6%
GLA, 0.3% DHA, 0.06% EPA; BO + FOHIGH: 0.6% GLA, 0.3%
DHA, 0.2% EPA] or human milk (HM, nonrandomized) for 4 wk.
ciable LC-PUFA concentrations is associated with the neuro-
logical development of preterm (3) and term babies (4,5).
This is ascribed to the importance of these fatty acids for
membrane properties, as there are high relative concentra-
tions of DHA in retina and brain (6). AA and other 20-carbon
atom fatty acids [dihomo-γ-linolenic (DGLA) and eicosapen-
taenoic (EPA)] are important precursors for eicosanoids (7).
Although functional effects of pure AA supplementation have
not been investigated in humans, a stable supply of AA, as
achieved by breast feeding (8), seems desirable for formula-
Anthropometric measures and blood samples were obtained at fed babies. In rats negative effects of low AA plasma concen-
study entry and after 14 and 28 d. There were no significant dif-
ferences between groups in anthropometric measures, tocoph-
erol, and retinol status at any of the studied time points. The AA
content of plasma phospholipids was similar between groups at
study start and decreased significantly until day 28 in all formula-
fed groups, but not in the breast-fed infants [ST: 6.6 0.2%, BO:
6.9 0.3%, BO + FOLOW: 6.9 0.4%, BO + FOHIGH: 6.7
0.2%, HM: 8.6 0.5%, where values are reported as mean
standard error; all formulas significantly different (P ≤ 0.05) from
HM]. There was no significant influence of GLA or fish oil addi-
tion to the diet. GLA had only a very limited effect on AA status
trations, induced by fish oil feeding, on postnatal develop-
ment have been shown (9). Furthermore, AA status has been
associated with infant growth (10,11).
An exogenous supply of LC-PUFA is required to achieve
plasma concentrations equivalent to those found in breast-fed
babies, although newborns already synthesize LC-PUFA en-
dogenously from the essential precursors linoleic (LA) and
α-linolenic acid during the first days after birth (12–14). Al-
though the pathways of conversion have been elucidated, the
question whether the low plasma and tissue concentrations
which was too small to obtain satisfactory concentrations (con- found without dietary supplement are due to the high meta-
centrations similar to breast-fed babies) under the circumstances
tested. The effect of GLA on AA is independent of the EPA and
DHA content in the diet within the dose ranges studied.
Paper no. L8539 in Lipids 36, 555–566 (June 2001).
bolic demand of LC-PUFA or to low conversion has not been
answered (15). The pathways of n-3 and n-6 essential fatty
acid conversion to AA and DHA share the enzymes for ∆-6,
∆-5 desaturation and chain elongation, but it is not yet known
whether there are different variants of these enzymes (16).
The initial step is a ∆-6 desaturation, which converts LA to γ-
The addition of suitable amounts of long-chain polyunsatu- linolenic acid (GLA). There are strong indications that this is
rated fatty acids (LC-PUFA) to infant formulas, especially ar- the rate-limiting step for the endogenous synthesis of AA
achidonic (AA) and docosahexaenoic (DHA) acids, estab- (17). Based on this concept, an exogenous supply of GLA
lishes concentrations of these fatty acids in infant plasma might increase the plasma concentration of n-6 LC-PUFA in
phospholipids equivalent to those found in breast-fed infants infants, as was reported to occur in adults (18).
Many manufacturers produce infant formulas containing
*To whom correspondence should be addressed at Division Metabolic Dis-
from 0.4 to 1.0% of fatty acids as LC-PUFA (19). Although
fish oils are readily available as a source for n-3 LC-PUFA,
the introduction of AA is a greater challenge. Egg phospho-
lipids and single-cell oils are the primary AA sources used in
infant formulas, but they are comparatively expensive or have
not been approved for formula use in all countries. Alterna-
tively, readily available sources of GLA-containing oils, e.g.,
borage, black currant seed or evening primrose oil, could be
orders & Nutrition, Department of Pediatrics, Dr. von Haunersches Kinder-
spital, University of Munich, Lindwurmstrasse 4, D-80336 München, Ger-
many. E-mail: berthold.koletzko@kk-i.med.uni-muenchen.de
Abbreviations: AA, arachidonic acid (20:4n-6); BO, borage oil; Chol, cho-
lesterol; DGLA, dihomo-γ-linolenic acid (20:3n-6); DHA, docosahexaenoic
acid (22:6n-3); EPA, eicosapentaenoic acid (20:5n-3); GLA, γ-linolenic acid
(18:3n-6); HM, human milk; LA, linoleic acid (18:2n-6); LC-PUFA, long-
chain polyunsaturated fatty acids (≥ 20 C atoms, ≥ 2 double bonds); MUFA,
monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; SAT, satu-
rated fatty acid; ST, standard formula; TG, triglycerides.
Copyright © 2001 by AOCS Press
555
Lipids, Vol. 36, no. 6 (2001)

556
H. DEMMELMAIR ET AL.
added to the formulas (20). These oils have been considered obtained for all infants before enrollment. If mothers provided
safe in older children even in pharmacological doses (21). HM, the infants were fed with breast milk. If mothers chose not
Some studies investigated the influence of a GLA and n-3 to provide breast milk, the infants were randomized double
LC-PUFA supply to term infants and found no positive ef- blind to one of the four formula groups without further stratifi-
fects on AA percentage in red blood cell phospholipids cation. Randomization numbers were computer-generated
(22–24). Since formulas tested in these studies also contained (Roche, Basel, Switzerland) and allocated sequentially in the
fish oils rich in EPA, it appeared possible that the lack of a order of enrollment to the infants. Double blinding was
positive effect of the GLA supplement might have been due achieved, as the identity of the dispensed formula was provided
to an inhibiting effect of high EPA and DHA supply on AA in a sealed envelope for each infant. Only in case of an immedi-
synthesis (8,20). This is based on the observation that feeding ately reportable adverse event was this envelope to be opened.
n-3 LC-PUFA-rich marine oil caused increased concentra-
The study formulas were produced by Nutricia (Zoetermeer,
tions of n-3 LC-PUFA and decreased AA concentrations in The Netherlands) based on the low birth weight infant formula
infants and adults (25,26). However, in term infants a formula “Nenatal.” The formulas delivered 80 kcal per 100 mL, and
containing 0.5% GLA only marginally increased the AA contained 11% (of energy) protein (whey/casein = 3:2), 40%
level, although EPA contributed only 0.07% to dietary fatty carbohydrates (mainly lactose and polysaccharides), and 49%
acids (22). DHA sources that contain only minor amounts of fat. The dietary fat (4.4 g/100 mL) was based on a blend of milk
EPA are now readily available, thus we investigated the in- fat, coconut oil, soy oil, sunflower oil, and canola oil. These
clusion of a slightly higher dose of GLA into a preterm for- were the only fat components of the standard formula (ST),
mula as an alternative to AA supplementation.
whereas the GLA content was increased by the addition of bor-
We compared four different formulas for preterm infants age oil (BO). The two other formulas (BO + FOLOW, BO +
containing: (i) no GLA and no LC-PUFA, (ii) 0.6% GLA and FOHIGH) additionally contained fish oils differing in their
no LC-PUFA, (iii) 0.6% GLA and 0.3% DHA combined with EPA/DHA ratio (Hoffmann-La Roche Ltd., Basel, Switzer-
0.06% EPA, and (iv) 0.2% EPA and 0.3% DHA plus 0.6% land). Both were composed to deliver 0.3% DHA but contained
GLA. A nonrandomized reference group of breast-fed infants either 0.06% (BO + FOLOW) or 0.2% EPA (BO + FOHIGH).
was also followed. While the majority of the previous infant Table 1 shows the detailed fatty acid composition of the differ-
studies analyzed red blood cells, which might have an attenu- ent formulas and of the breast milk fed (mean and standard
ated and delayed response to dietary changes, we chose to an- error of individual samples), as analyzed in our laboratory. In
alyze plasma phospholipids.
the case of breast milk feeding, a 5-mL sample of breast milk
Since LC-PUFA supplementation of the diet decreased the was collected on days 0 and 14. All formulas contained 0.1 mg
tocopherol/lipid ratio in the erythrocytes of infants and the vitamin A and 1.3 mg vitamin E per 100 mL.
plasma vitamin E concentrations in adults (27,28) and since in
On the day of enrollment (study day 0), 2 wk later (study
newborns serious health risks have been associated with low vi- day 14), and 4 wk later (study day 28) anthropometric measures
tamin E levels (29), we also analyzed plasma concentrations of were recorded and blood samples were taken. At all time points
lipid-soluble vitamins to detect potential influences of the vari- a deviation of maximally 1 d from the date determined by the
ous fatty acid supplies on the infants’ antioxidative status (30).
protocol was tolerated. Weight, length, and head circumference
were measured according to standard hospital procedures.
Blood samples (0.5 mL) were taken by venipuncture and im-
mediately transferred into EDTA-containing tubes. Blood cells
SUBJECTS AND METHODS
Premature infants were recruited for the study at the Chil- and plasma were separated by centrifugation at 1000 × g for 5
dren’s Hospital Gyor (Hungary) from December 1994 to May min. A plasma aliquot of at least 200 µL was frozen immedi-
1997. Inclusion criteria were a birth weight below 1800 g and ately at −80°C for later analysis. Red blood cells were washed
full enteral feeding with more than 120 mL milk/kg/d. Ex- three times in 0.9% aqueous NaCl solution, hemolyzed, and
cluded from the study were any infants with serious metabolic stored at −80°C until analysis (data not shown). The remaining
or congenital anomalies; infants on artificial ventilation; in- portion of the sample was used for the determination of routine
fants with septic infections, gastrointestinal problems or laboratory parameters: alanine-amino-transferase, γ-glutamyl-
metabolic diseases; and infants receiving any parenteral lipids transpeptidase, total bilirubin, creatinine, total protein, glucose,
during the study period. Systemic corticosteroid therapy dur- carbamide-N, sodium, potassium, and calcium. For further as-
ing the study was only allowed for up to 3 d, otherwise sub- sessment of possible adverse effects a series of hematological
jects were excluded. According to the standard hospital pro- measures were performed, e.g., red blood cell count, hemoglo-
tocol for parenteral nutrition, all participating infants were bin, hematocrit, mean corpuscular volume, hemoglobin E,
fed a 20% lipid emulsion based on soybean oil. Up to 10% of white blood cell count, platelet count, and hemogram. Pulse
total energy intake from sources other than the corresponding rate and blood pressure were also recorded.
study diet were permitted.
As differences of 20% or greater in the primary study pa-
The study protocol was approved by the Ethical Committee rameter, AA concentration in plasma phospholipids, were
of the Hungarian Medical Association. After explanation of the considered clinically important, the sample size was planned
study protocol, written parental consent for participation was on the data of Decsi and Koletzko (31) who reported a rela-
Lipids, Vol. 36, no. 6 (2001)

BORAGE OIL AND PLASMA ARACHIDONIC ACID IN PREMATURE INFANTS
557
TABLE 1
Fatty Acid Composition (%w/w) of the Formulas Used in the Study (results of our own analyses)
and from the Individual Human Milk Samples on Day 0 and Day 14a
HM
BO
(n = 4)
Day 0
(n = 12)
Day 14
(n = 13)
ST
BO + FOLOW BO + FOHIGH
Fatty acid
(n = 2)
(mean SE)
(n = 2)
(n = 2)
(mean SE)
(mean SE)
∑SAT
41.10/41.34 41.53 0.07 40.87/40.96
44.05/43.88 43.75 0.11 44.02/44.09
40.44/40.64
44.07/43.88
0.07/0.07
ND
46.32 1.76
34.68 1.12
0.72 0.14
0.01 0.01
47.47 1.17
34.19 1.09
0.85 0.14
0.01 0.01
∑MUFA
∑TRANS
20:3n-9
0.05/0.05
ND
0.03 0.01
ND
0.05/0.06
ND
18:2n-6
18:3n-6
20:2n-6
20:3n-6
20:4n-6
22:2n-6
22:4n-6
13.47/13.40 12.88 0.03 12.73/12.74
12.97/12.97
0.64/0.64
0.02/ND
0.01/0.01
0.02/0.02
0.05/0.05
ND
15.65 1.25
0.15 0.01
0.40 0.04
0.43 0.03
0.51 0.03
0.05 0.01
0.14 0.01
15.15 1.14
0.17 0.01
0.32 0.03
0.41 0.03
0.47 0.03
0.04 0.01
0.12 0.01
0.01/0.01
ND
0.65 0.02
0.01 0.00
0.01 0.00
0.01 0.00
0.06 0.00
ND
0.60/0.60
0.01/0.01
0.01/0.01
0.03/0.03
0.06/0.06
ND
0.01/0.01
0.01/0.01
0.06/0.06
ND
18:3n-3
18:4n-3
20:3n-3
20:5n-3
22:5n-3
22:6n-3
1.21/1.20
0.02/0.02
ND
0.01/0.01
0.01/0.01
ND
1.06 0.09
0.02 0.01
ND
ND
0.01 0.00
ND
1.20/1.20
0.02/0.02
ND
0.06/0.06
0.02/0.02
0.24/0.25
1.26/1.26
0.04/0.04
ND
0.15/0.15
0.02/0.02
0.25/0.25
0.51 0.05
0.03 0.01
0.02 0.01
0.03 0.01
0.11 0.01
0.22 0.03
0.43 0.04
0.03 0.01
0.01 0.01
0.02 0.01
0.10 0.01
0.20 0.03
LC-PUFA
0.10/0.10
0.10 0.00
0.42/0.42
0.52/0.51
1.95 0.12
1.73 0.12
18:2n-6/18:3n-3 11.10/11.12 12.39 1.06 10.61/10.63
10.30/10.33
33.92 3.87
38.64 4.56
aST, standard formula; BO, borage oil; BO + FOLOW, borage oil + 0.3% DHA and 0.06% EPA; BO + FOHIGH, borage oil
+ 0.3% DHA and 0.2% EPA; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; SAT, saturated fatty acids; MUFA,
monounsaturated fatty acids; TRANS, trans fatty acids; ND, not detected; LC-PUFA, long-chain polyunsaturated fatty acids.
tive standard deviation of 24% for AA. With 13 infants per detector (300°C). Separation of individual fatty acid methyl es-
group, the considered difference between groups was to be ters was achieved with a BPX70 column (SGE, Weiterstadt,
detected with 90% probability and a 5% risk of type I error.
Germany) with 60 m length and 0.32 mm inner diameter. The
Analysis of plasma phospholipids. After the addition of temperature program started at 130°C, increased at a rate of
dipentadecanoyl-phosphatidylcholine as an internal standard, 3°C/min up to 200°C, and continued without delay up to 210°C
total lipids from 100 µL plasma were extracted into 2 mL at a rate of 1°C/min. For identification and determination of re-
hexane/isopropanol (4:1) and subsequently twice into 2 mL of sponse factors, commercially available standards were used
hexane (32). Extracts were combined and dried under a stream [Nu-Chek-Prep, Elysian, MN; Sigma, Deisenhofen, Germany;
of N2. For application on silica gel plates (Merck KG, Darm- docosapentaenoic (n-6) acid methyl ester was a gift from
stadt, Germany), the residue was taken up in chloroform/ Omega Tech, Boulder, CO]. Chromatographic data were
methanol (1:1). Phospholipids were isolated by development recorded and evaluated using EZChrom Elite Ver. 2.1 software
of the plates in n-heptane/diisopropylether/glacial acetic acid (Scientific Software Inc., Pleasanton, CA). Because of the
(60:40:3, by vol) (33). After visualization with 2,7-dichloroflu- small plasma volumes, samples were weighed in addition to
orescein, phospholipid-containing bands were scraped off and volumetric dosage, and absolute plasma concentrations are
transferred into reaction vials. Synthesis of fatty acid methyl given as mg fatty acid methyl ester per kg plasma.
esters was performed by heating the phospholipids to 85°C for
Analysis of milk lipids. Milk samples were analyzed as pre-
45 min after dissolution in 1.5 mL 3M methanolic hydrochlo- viously described (34). Briefly, after the addition of 100 µL
ric acid. After neutralization of the reaction mixture with car- potassium oxalate (3.5%) solution, lipids from 1 mL milk
bonate buffer, methyl esters were extracted twice into 1 mL were extracted into 3 mL of a mixture of ethanol/tert-butyl-
hexane and the combined extracts taken to dryness under nitro- methylether/petrolether (1:1:1). To obtain quantitative recov-
gen. For storage (−80°C) until gas chromatographic analysis, ery, a second extraction with 2 mL tert-butyl-methylether/
the samples were dissolved in 40 µL hexane (containing 2 g/L petrolether (1:1) was performed. Extracts were combined and
butylhydroxytoluene) and transferred into microvials.
transferred into a preweighed vial; solvents were evaporated
Gas chromatography was performed on an HP5890 series II under nitrogen, and the remainder was dried in a desiccator
gas chromatograph (Hewlett-Packard, Waldbronn, Germany), overnight. Total lipid content could now be determined gravi-
equipped with a split/splitless injector (250°C, split ratio = metrically. For transesterification at 90°C for 60 min, the lipids
1:20, column head pressure 1.20 bar) and a flame-ionization were dissolved in 2 mL 1.5 M methanolic hydrochloric acid
Lipids, Vol. 36, no. 6 (2001)

558
H. DEMMELMAIR ET AL.
and 1 mL hexane. After the addition of 2 mL water, the methyl sponding values at study start as covariates. Differences be-
esters were extracted into 2 mL of hexane and analyzed by gas tween group means were taken as statistically significant if the
chromatography as described for plasma samples.
95% confidence interval (Scheffé method for correction of
Analyses of vitamins A and E. These analyses were per- multiple testing) of differences between groups did not include
formed as previously described from 100 µL plasma samples 0. Coefficients of correlation were calculated according to
by high-performance liquid chromatography (35). After the Pearson and were taken as significant if P was equal to or less
addition of 100 µL ethanol to the samples, vitamins were ex- than 0.05. Individual changes with time were investigated by
tracted three times into 1 mL hexane and the combined ex- paired t-tests (significance level 0.05). Calculations were per-
tracts taken to dryness. For chromatography the extract was formed using S Plus, Ver. 4.5 (Mathsoft Inc., Seattle, WA).
reconstituted in 100 µL mobile phase [acetonitrile/tetrahydro-
furan/methanol/ammonium acetate (1%) = 684:220:68:28]
containing tocol as standard for quantification. A high-perfor-
RESULTS
mance liquid chromatography system (L6200 pump, AS 2000 Sixty-six infants with gestational ages between 25 and 37 wk
autosampler, L4250 ultraviolet-visible detector, F-1050 fluo- were enrolled into the study. One infant from group ST was
rescence detector; Merck), equipped with a reversed-phase excluded from the study because of infectious disease. Thus
column (LiChrospher 100, RP-18, 25 cm long, Merck) was the results from 65 infants who completed the study were
used for chromatography. With a flow rate of 0.65 mL/min, evaluated. There were no statistically significant differences
retinol, α-tocopherol and δ-tocopherol could be separated, between the five dietary groups in clinical characteristics,
while β- and γ-tocopherol coeluted. For retinol detection the body weight, length, head circumference, and postnatal age
photometer was set to 325 nm, and tocopherols were detected (Table 2). Although infants in groups BO and BO + FOHIGH
by their fluorescence at 330 nm after excitation at 298 nm. tended to be older, there were no significant differences at
Data were recorded and evaluated with EZChrom Elite Ver. study start and study end.
2.1 software (Scientific Software Inc., Pleasanton, CA).
Infants presented very similar fatty acid compositions of
Total triglycerides (TG) and total cholesterol (Chol) were plasma phospholipids across all groups at study start
determined using a Vitros 250 autoanalyzer (Johnson & John- (Table 3). Saturated fatty acids (SAT) contributed almost one-
son, Neckargmünd, Germany).
half and cis-monounsaturated about 20% of the total. The
Statistical analysis. Data characterizing subjects are given only major difference at study start was a lower GLA content
as means standard deviations. One-way analysis of variance in the plasma phospholipids of the breast-fed group, although
was used to detect statistically significant differences (P ≤ 0.05) 0.15% GLA was detected in HM fat.
between groups. Analytical results for fatty acids and lipid-sol-
Tendencies indicating the statistically analyzed values at
uble vitamins are presented as means standard errors of the study end could already be observed for all investigated
mean. Statistical evaluation of the results at the end of the study parameters at study day 14; thus there seemed to be a contin-
was performed by analysis of variance, including the corre- uous trend during the whole study period.
TABLE 2
Clinical and Anthropometric Description of the Infants (mean SD) Studied at Birth,
Day 0 (study start), Day 14, and Day 28 (study end)
ST
BO
BO + FOLOW BO + FOHIGH
HM
Number of infants
Birth weight (g)
Age at study start (d)
13
13
13
14
13
1494 269
21.2 10.4
1302 229.3
32.4 18.2
1500 199
22.6 10.9
1362 284
33.9 15.1
1404 290
29.5 13.3
Body weighta (g)
Day 0
Day 14
Day 28
1522 222
1840 275
2315 325*
1422 188
1790 249
2239 306*
1535 198
1888 223
2388 250*
1516 223
1865 319
2391 377*
1532 250
1822 294
2239 380*
Lengtha (cm)
Day 0
Day 14
Day 28
43.2 2.9
44.0 2.4
46.6 2.9*
42.2 2.1
44.2 1.9
46.0 2.3*
42.9 3.4
44.1 2.7
46.9 1.7*
42.8 1.7
44.6 1.9
46.2 1.9*
42.7 2.5
44.0 2.2
46.3 2.5*
Head circumferencea (cm)
Day 0
Day 14
Day 28
30.0 2.0
31.3 1.8
32.9 1.5*
28.2 2.8
30.7 1.7
32.2 1.5*
29.2 1.7
31.5 1.7
33.0 1.1*
29.9 1.6
31.5 1.7
32.86 1.6*
28.9 2.0
30.6 1.1
32.2 0.9*
aAsterisk indicates a significant change (P ≤ 0.05) from day 0 to day 28. For abbreviations for formulas used see Table 1.
Lipids, Vol. 36, no. 6 (2001)

BORAGE OIL AND PLASMA ARACHIDONIC ACID IN PREMATURE INFANTS
559
TABLE 3
Fatty Acid Composition (%w/w, mean SE) of Infant Plasma Phospholipids at Study Start (day 0),
After 2 wk on Study Diet (day 14), and at Study End After 4 wk (day 28)
ST
BO
BO + FOLOW
BO + FOHIGH
HM
Day 0
∑SAT
48.01 0.87
20.69 0.78
0.86 0.07
0.78 0.22
13.94 0.94
0.11 0.03
0.39 0.04
2.71 0.17
9.37 0.52
0.55 0.04
0.50 0.05
0.30 0.02
13.51 0.62
ND
0.06 0.03
0.19 0.04
1.85 0.09
2.10 0.13
30.43 1.02
0.64 0.03
16.39 0.72
47.53 0.53
21.64 1.13
0.89 0.06
0.96 0.31
13.73 1.20
0.12 0.02
0.47 0.03
2.68 0.12
8.55 0.41
0.50 0.04
0.55 0.03
0.32 0.02
12.74 0.50
0.03 0.01
0.15 0.02
0.27 0.05
1.93 0.14
2.35 0.18
29.94 1 .40
0.63 0.03
16.05 0.46
47.72 0.71
20.11 0.78
0.83 0.07
0.48 0.09
14.49 0.90
0.10 0.03
0.44 0.02
2.69 0.11
9.80 0.36
0.49 0.04
0.53 0.03
0.28 0.02
13.95 0.35
0.01 0.01
0.14 0.05
0.18 0.04
1.97 0.08
2.30 0.12
31.33 0.80
0.66 0.02
16.72 0.40
48.21 0.32
19.61 0.86
0.87 0.04
0.81 0.21
14.57 1.08
0.12 0.03
0.46 0.02
2.83 0.17
9.18 0.42
0.56 0.05
0.55 0.03
0.32 0.03
13.57 0.49
0.02 0.01
0.09 0.04
0.25 0.04
1.87 0.10
2.22 0.13
31.30 1.06
0.65 0.02
16.60 0.51
48.28 0.46
19.44 0.45
0.94 0.07
0.47 0.15
14.72 0.44
0.01 0.01
0.46 0.02
2.83 0.17
9.47 0.35
0.59 0.04
0.51 0.02
0.30 0.02
13.85 0.42
ND
0.04 0.02
0.24 0.03
2.01 0.14
2.29 0.16
31.33 0.71
0.65 0.02
16.61 0.54
∑MUFA
∑TRANS
20:3n-9
18:2n-6
18:3n-6
20:2n-6
20:3n-6
20:4n-6
22:2n-6
22:4n-6
20:3/20:4
∑n-6 LC-PUFA
18:3n-3
20:5n-3
22:5n-3
22:6n-3
∑n-3 LC-PUFA
∑PUFA
∑PUFA/∑SAT
∑LC-PUFA
Day 14
∑SAT
43.86 0.44
23.77 0.37
0.55 0.03
0.19 0.04
18.38 0.60
0.06 0.02
0.42 0.02
2.51 0.17
7.37 0.38
0.52 0.05
0.36 0.03
0.35 0.03
11.18 0.42
0.05 0.02
0.14 0.03
0.16 0.03
1.66 0.13
1.95 0.15
31.82 0.26
0.73 0.01
13.33 0.52
43.91 0.46
23.60 0.38
0.54 0.03
0.20 0.03
17.81 0.53
0.15 0.02
0.43 0.02
3.17 0.11
7.33 0.22
0.52 0.05
0.38 0.02
0.44 0.02
11.83 0.22
0.09 0.02
0.13 0.04
0.20 0.03
1.55 0.12
1.87 0.15
31.96 0.48
0.73 0.02
13.91 0.28
44.36 0.68
23.33 0.56
0.52 0.02
0.17 0.03
16.59 0.38
0.16 0.02
0.40 0.02
3.25 0.18
7.57 0.24
0.51 0.06
0.32 0.03
0.43 0.03
12.05 0.26
0.03 0.01
0.36 0.02
0.16 0.04
2.28 0.12
2.80 0.14
31.79 0.44
0.72 0.02
15.01 0.40
44.56 0.46
22.99 0.54
0.53 0.05
0.13 0.04
16.93 0.38
0.13 0.02
0.41 0.02
3.06 0.11
7.43 0.28
0.63 0.04
0.32 0.03
0.42 0.03
11.85 0.24
0.05 0.02
0.42 0.04
0.22 0.02
2.17 0.09
2.82 0.11
31.91 0.28
0.72 0.01
14.80 0.26
47.98 0.52
18.94 0.43
0.97 0.05
0.39 0.11
16.31 0.60
0.08 0.03
0.49 0.02
2.88 0.14
8.57 0.33
0.57 0.03
0.54 0.03
0.34 0.02
13.05 0.37
0.01 0.01
0.08 0.03
0.32 0.02
1.89 0.14
2.29 0.15
32.11 0.68
0.67 0.02
15.72 0.44
∑MUFA
∑TRANS
20:3n-9
18:2n-6
18:3n-6
20:2n-6
20:3n-6
20:4n-6
22:2n-6
22:4n-6
20:3/20:4
∑n-6 LC-PUFA
18:3n-3
20:5n-3
22:5n-3
22:6n-3
∑n-3 LC-PUFA
∑PUFA
∑PUFA/SAT
∑LC-PUFA
Day 28
∑SAT
44.08 0.45a,*
24.00 0.42a,*
0.49 0.06a,*
0.29 0.03*
18.66 0.34a,*
0.09 0.02
43.95 0.47b,*
23.63 0.31b
0.54 0.03b,*
0.16 0.02a,*
18.41 0.34*
0.15 0.01
40.95 3.45c,*
21.35 1.87c,*
0.54 0.07c,*
0.19 0.03*
17.31 0.51*
0.15 0.03
44.52 0.40d,*
23.05 0.43d,*
0.58 0.04d,*
0.14 0.03b,*
17.64 0.27*
0.14 0.02
47.54 0.63a,b,c,d
19.30 0.63a,b,c,d
0.90 0.06a,b,c,d
0.50 0.15a,b
16.51 0.84a,*
0.09 0.03*
∑MUFA
∑TRANS
20:3n-9
18:2n-6
18:3n-6
20:2n-6
0.45 0.02
0.42 0.02
0.44 0.02
0.39 0.02*
3.00 0.09
0.46 0.02
20:3n-6
2.69 0.13
3.11 0.17
3.29 0.18*
6.87 0.38c,*
0.48 0.04
2.86 0.19
20:4n-6
6.64 0.21a,*
0.59 0.05
6.94 0.27b,*
0.58 0.05
6.71 0.20d,*
0.62 0.05
8.57 0.47a,b,c,d
0.63 0.04
22:2n-6
22:4n-6
0.31 0.01a,*
0.41 0.02*
10.69 0.27a,*
0.05 0.02*
0.12 0.03a
0.15 0.03a,b
1.37 0.07a,b,
1.64 0.09a,b,
31.42 0.28
0.71 0.01*
12.62 0.35a,*
0.35 0.03b,*
0.46 0.03*
11.40 0.32*
0.10 0.02a,*
0.13 0.04b,c
0.20 0.03c
1.33 0.13c,d,
1.66 0.16c,d,
31.87 0.45
0.73 0.02*
13.22 0.42*
0.32 0.03c,*
0.50 0.04a,*
10.52 0.95b,*
0.06 0.02*
0.35 0.06b,d,
0.26 0.02a,*
0.25 0.02d,*
0.45 0.02*
10.99 0.23c,*
0.06 0.02*
0.41 0.05a,c,e,
0.20 0.03d
0.52 0.04a,b,c,d
0.34 0.02a
20:3/20:4
∑n-6 LC-PUFA
18:3n-3
13.05 0.55a,b,c
0.01 0.01a
20:5n-3
*
*
0.06 0.03d,e
22:5n-3
0.32 0.02b,c,d,
*
22:6n-3
*
*
*
*
2.20 0.12a,c,e
2.60 0.27a,c,e,
29.48 2.50
2.27 0.09b,d,f,
2.88 0.13b,d,f,
31.85 0.22
*
*
1.72 0.14e,f,
2.10 0.16e,f
32.26 0.48
0.68 0.02
*
∑n-3 LC-PUFA
*
∑PUFA
∑PUFA/∑SAT
∑LC-PUFA
0.72 0.02*
13.30 1.20*
0.72 0.01*
14.01 0.27*
15.65 0.68a
aCommon roman letter superscripts within a row indicate a significant difference (P ≤ 0.05) between groups. For abbreviations see Table 1.
Lipids, Vol. 36, no. 6 (2001)

560
H. DEMMELMAIR ET AL.
After 4 wk on the study diet, the percentage of SAT fatty
acids decreased, while cis-monounsaturated fatty acids
(MUFA) increased in all formula groups (Tables 3,4). In con-
trast, there were no changes in the breast-fed group, reflecting
a higher percentage of SAT and a lower percentage of cis-
MUFA in breast milk compared to the formula diets (Tables
3,4). The trans fatty acid content in the plasma lipids of breast-
fed babies was almost twice as high as in all formula-fed
groups, which corresponds to about 0.8% trans fatty acids in
breast milk and no detectable trans-MUFA in formulas based
on plant oil. Mead acid (20:3n-9) percentage decreased signif-
icantly over time in all formula groups, but not in HM-fed in-
fants. In the diet, the percentage of the precursor oleic acid was
higher in the formulas than in breast milk (43 vs. 31%), but
traces of Mead acid were detectable only in breast milk.
At day 28, on the diets with 0.6% GLA there was no
marked difference in phospholipid GLA content (0.1% in
group ST vs. 0.15% in the other formula groups, not signifi-
cant); only the breast-fed group showed a significant increase
with time. No obvious difference was caused by additional
dietary supply of n-3 LC-PUFA in the diet. The same trend
was observed for the absolute concentrations, with all mean
values below 2.5 mg/kg plasma. There were no significant
FIG. 1. Individual α-tocopherol concentrations (µmol/L) in relation to
the percentage of LC-PUFA in plasma phospholipids (ꢀ ST, ꢁ BO, ꢂ
BO + FOLOW, ꢃ BO + FOHIGH, ꢄ HM) showing neither a significant
difference between groups nor a significant correlation for any of the
groups. Abbreviations: LC-PUFA, long-chain polyunsaturated fatty acids;
ST, standard formula; BO, borage oil; BO + FOLOW, borage oil + 0.3%
docosahexaenoic acid (DHA) and 0.06% eicosapentaenoic acid (EPA);
BO + FOHIGH, BO + 0.3% DHA + 0.2% EPA; HM, human milk.
differences in the content (absolute and relative) of DGLA, of n-6 LC-PUFA to n-3 LC-PUFA. The ratios at day 28 were
which is obtained by chain elongation of GLA. LA content similar in breast-fed infants and in infants from groups ST
increased significantly with time in all groups, but only the and BO, whereas the n-6/n-3 LC-PUFA ratio dropped signifi-
differences between groups ST and HM evolved to signifi- cantly (P ≤ 0.05) from 6.27 0.33 (M SE) to 4.23 0.30 in
cance at day 28. The concentration of the elongation product BO + FOLOW and from 6.36 0.36 to 3.97 0.27 in BO +
of LA, 20:2n-6, like LA, showed no significant group differ- FOHIGH, respectively, and in BO it increased significantly
ences, although only HM contained 20:2n-6. The content of from 5.72 0.39 to 7.36 0.48.
the major n-6 LC-PUFA AA, which was very similar in all
The amount of antioxidative vitamins in the formulas (0.1
groups on day 0, decreased significantly until day 28 in all mg retinol and 1.3 mg tocopherol per 100 mL) resulted in
formula groups but not in the breast milk (0.5% AA) group, plasma concentrations of retinol and α-tocopherol that were
indicating that supplementation with GLA did not increase not different from breast-fed infants (Table 5). There was no
the AA content (Table 3). After 4 wk on the study diets, the detectable influence of LC-PUFA addition to the formulas on
AA percentage was significantly higher in the HM group, this parameter. In addition, there was no significant correla-
whereas no other group differences approached statistical sig- tion between LC-PUFA percentage and α-tocopherol concen-
nificance. Absolute concentrations were only different be- tration (Fig. 1). Only for δ-tocopherol, which showed low
tween ST and HM (Table 4). The chain elongation product of concentrations in all infants and which is an isomer with low
AA (22:4n-6) was significantly lower in the formula-fed biological activity (36), could significant differences be ob-
groups than in HM, but this cannot be ascribed to elongase served between BO and HM as well as BO + FOHIGH and
activity since only breast milk contained 22:4n-6 (0.14%).
HM. The adjustment of α-tocopherol and retinol concentra-
Addition of fish oil that was either high or low in EPA con- tions for Chol and TG by multiple regression according to
tent did not further decrease n-6 LC-PUFA but markedly in- Jordan et al. (37) did not reveal significant differences be-
creased the DHA content in phospholipids (Tables 3,4). Mean tween groups (Table 5). In all groups there was a trend for α-
EPA percentage was about twice as high in these groups as in tocopherol to increase with time, but it reached significance
all other groups, although EPA contributed only 0.06 and at day 28 only in groups BO and BO + FOHIGH.
0.15% to dietary fatty acids. Absolute concentrations of EPA
were more than sixfold higher in the fish oil groups than in the
HM group and twofold higher than in the other formula groups,
DISCUSSION
respectively (Table 4). In contrast to EPA and DHA, the levels This study was designed to evaluate the influence of added
of the intermediate 22:5n-3 tended to be higher in HM infants, GLA in preterm infant formula on the composition of n-6 LC-
who received an exogenous supply (0.1%) of 22:5n-3. There PUFA in infant plasma phospholipids. The added GLA re-
was no significant difference between groups BO + FOLOW sulted in dietary intakes of at least 32–34 mg/kg/d in groups
and BO + FOHIGH in any fatty acid concentration.
BO, BO + FOLOW and BO + FOHIGH, which is close to the
There was a marked influence of fish oil supply on the ratio sum of GLA, DGLA, and AA typically ingested with breast
Lipids, Vol. 36, no. 6 (2001)

BORAGE OIL AND PLASMA ARACHIDONIC ACID IN PREMATURE INFANTS
561
TABLE 4
Absolute Concentrations of Fatty Acids in Plasma Phospholipids (mg/kg plasma, mean SE) for the Three Time Points Investigateda
ST
BO
BO + FOLOW
BO + FOHIGH
HM
Day 0
Total fatty acids
∑SAT
1042.7 59.7
500.1 28.6
217.6 17.7
9.0 0.9
9.2 2.9
141.0 7.7
1.2 0.4
1171.5 51.8
555.7 22.9
253.2 16.1
10.3 0.7
11.2 3.7
161.9 17.1
1.4 0.3
1113.5 41.2
530.9 20.2
222.3 8.7
9.1 0.8
5.3 1.0
164.0 15.3
1.1 0.3
1029.9 43.4
496.3 20.4
201.0 10.8
9.0 0.7
8.3 2.3
150.9 14.0
1.2 0.3
963.2 46.7
465.0 23.3
186.4 9.0
9.0 0.6
4.9 1.8
141.2 6.8
0.1 0.1
∑MUFA
∑TRANS
20:3n-9
18:2n-6
18:3n-6
20:2n-6
20:3n-6
20:4n-6
22:2n-6
4.1 0.4
5.5 0.5
4.9 0.4
4.7 0.2
4.4 0.3
28.5 2.5
98.9 9.3
5.7 0.5
31.3 2.0
100.7 7.6
5.7 0.4
29.9 1.6
108.9 5.4
5.4 0.4
29.2 2.3
94.8 6.3
5.7 0.6
27.4 2.3
92.1 6.4
5.6 0.4
22:4n-6
∑n-6 LC-PUFA
18:3n-3
5.4 0.7
142.6 12.4
ND
6.4 0.5
149.7 10.0
0.4 0.2
5.9 0.4
155.1 6.6
0.1 0.1
5.6 0.6
140.1 8.3
0.2 0.1
4.9 0.3
134.3 8.6
ND
20:5n-3
22:5n-3
22:6n-3
0.7 0.3
1.9 0.4
19.5 1.7
22.0 2.0
316.0 19.5
173.8 15.4
1.8 0.3
3.2 0.6
22.8 2.2
27.8 2.7
352.3 25.8
188.7 11.3
1.5 0.5
0.9 0.36
2.6 0.4
19.5 1.6
23.1 2.0
323.7 19.9
171.4 9.8
0.4 0.2
2.4 0.3
19.5 1.8
22.3 2.0
302.8 16.7
161.5 11.0
2.03 0.5
22.0 1.2
25.5 1.6
351.0 19.7
185.8 7.6
∑n-3 LC-PUFA
∑PUFA
∑LC-PUFA
Day 14
Total fatty acids
∑SAT
1142.7 68.9
501.2 30.6
271.7 17.0
6.4 0.7
1210.2 65.8
530.2 27.6
286.9 18.2
6.5 0.4
1136.4 54.4
503.5 24.7
264.5 12.8
5.9 0.4
1124.3 43.4
501.9 22.3
257.1 8.7
6.126 0.7
1.5 0.5
1063.7 66.6
509.9 32.6
201.8 14.4
10.4 0.9
4.5 1.8
∑MUFA
∑TRANS
20:3n-9
2.4 0.5
2.5 0.4
2.0 0.4
18:2n-6
18:3n-6
207.0 9.8
0.7 0.2
216.1 13.9
1.8 0.3
188.8 11.8
1.7 0.3
190.8 9.4
1.5 0.3
171.8 10.1
0.9 0.4
20:2n-6
20:3n-6
20:4n-6
22:2n-6
4.8 0.4
5.2 0.4
4.6 0.3
4.6 0.3
5.2 0.4
28.8 2.6
86.4 9.2
5.83 0.6
4.3 0.7
38.5 2.7
88.1 4.5
6.1 0.5
36.7 2.4
87.0 6.1
5.6 0.5
34.3 1.6
83.7 4.8
7.2 0.6
30.6 2.6
92.0 7.2
6.2 0.6
22:4n-6
4.5 0.3
3.8 0.4
3.6 0.3
5.7 0.5
∑n-6 LC-PUFA
18:3n-3
130.2 12.0
0.6 0.2
142.3 7.0
1.1 0.2
137.6 7.9
0.4 0.2
133.3 6.0
0.6 0.2
139.7 10.5
0.1 0.1
20:5n-3
22:5n-3
1.6 0.4
1.8 0.3
1.7 0.6
2.6 0.4
4.0 0.3
1.8 0.4
4.7 0.5
2.5 0.2
0.9 0.4
3.4 0.2
22:6n-3
19.2 1.9
22.6 2.3
363.4 21.8
155.2 1.0
18.7 1.9
22.9 2.4
386.69 21.6
167.74 8.9
26.3 2.2
32.1 2.6
362.6 20.0
171.6 10.5
24.2 1.0
31.4 1.3
359.1 14.7
166.2 6.8
20.4 2.2
24.7 2.5
341.7 21.6
168.9 13.4
∑n-3 LC-PUFA
∑PUFA
∑LC-PUFA
Day 28
Total fatty acids
∑SAT
1139.0 44.1
501.4 18.3
274.2 13.2*
5.7 0.8a,*
3.4 0.4
212.3 8.9*
1.1 0.3
1195.9 65.0
523.7 26.3
282.8 15.6
6.4 0.4b,*
2.0 0.4a,*
221.4 14.6*
1.8 0.2
1202.2 47.2
532.5 20.1*
279.3 15.2*
6.9 0.6*
2.4 0.5*
208.0 9.5*
1.9 0.4
1154.0 57.0
514.3 26.7*
265.1 12.3*
6.8 0.7c,*
1.6 0.3b,*
203.2 9.6*
1.7 0.2
1077.1 67.7
510.2 29.7*
209.7 18.0
∑MUFA
∑TRANS
20:3n-9
18:2n-6
9.6 0.7a,b,c
5.7 1.9a,b
176.3 12.3*
1.0 0.3*
18:3n-6
20:2n-6
20:3n-6
20:4n-6
22:2n-6
22:4n-6
∑n-6 LC-PUFA
18:3n-3
20:5n-3
22:5n-3
22:6n-3
5.1 0.3*
5.1 0.4
37.8 3.5
82.5 4.7
5.3 0.3
4.5 0.2
4.9 0.3
30.5 1.7
39.7 3.1*
82.1 4.7*
5.7 0.5
34.8 2.2*
77.7 5.0*
7.4 0.8*
30.4 2.5
75.6 3.8a,*
6.7 0.6
93.8 10.2a
6.8 0.6*
6.8 0.6
3.6 0.2a,*
121.5 5.3
0.6 0.2*
4.2 0.4b,*
136.5 8.3
1.2 0.2*
3.9 0.3c,*
136.7 6.7*
0.8 0.2*
3.0 0.4d,*
127.4 7.9
0.8 0.2*
5.6 0.6a,b,c,d
141.6 12.5
0.1 0.1
1.4 0.4a,b
1.8 0.4a,b
15.7 1.1a,b
18.9 1.4a,b
357.7 14.0
143.7 6.9
1.7 0.5c,d
2.5 0.4
4.2 0.8a,c,e,
3.2 0.2a,*
*
*
4.6 0.5b,d,f,
2.3 0.3
*
0.7 0.4e,f
3.4 0.3b,*
18.7 2.1e
22.8 2.7e,f
347.5 22.8*
170.1 15.4
16.1 1.8c,d,
20.2 2.4c,d,
383.0 24.1
158.7 10.4
*
*
26.5 1.9a,c,
*
26.3 1.8b,d,e,
*
∑n-3 LC-PUFA
33.9 2.6a,c,e,
383.6 15.6
172.9 9.1
33.3 2.2b,d,f,
367.9 18.9
162.3 9.8
*
∑PUFA
∑LC-PUFA
aAsterisk indicates a significant change (P ≤ 0.05) from Day 0 to Day 28; common roman letter superscripts within a row indicate a significant difference (P ≤
0.05) between groups. For abbreviations see Table 1.
Lipids, Vol. 36, no. 6 (2001)

562
H. DEMMELMAIR ET AL.
TABLE 5
Concentration (µmol/L, mean SE) of Retinol and Tocopherol Isomers in Infant Plasma at Study Start (day 0),
After 2 wk on Study Diets (day 14), and After 4 wk on Study Diets (day 28)a
ST
BO
BO + FOLOW
BO + FOHIGH
HM
Day 0
Retinol
0.49 0.08
13.03 1.52
0.11 0.03
0.61 0.18
13.78 1.33
0.51 0.06
101.3 9.3
81.3 8.9
0.38 0.03
12.08 1.86
0.09 0.02
0.59 0.14
11.13 1.41
0.37 0.03
112.5 8.6
116.6 17.4
0.39 0.05
12.18 1.66
0.1 0.02
0.48 0.11
12.88 1.63
0.41 0.05
101.9 5.2
82.0 8.6
0.36 0.04
14.48 1.92
0.11 0.03
0.67 0.21
15.20 1.94
0.41 0.04
89.5 6.6
0.43 0.05
13.80 1.90
0.08 0.01
0.49 0.12
14.18 1.87
0.43 0.05
104.7 11.2
86.8 7.4
α-Tocopherol
δ-Tocopherol
β,γ-Tocopherol
α-Tocopherol adj.
Retinol adj.
Chol (mg/dL)
TG (mg/dL)
78.6 7.2
Day 14
Retinol
0.34 0.03
18.15 3.7
0.27 0.06
1.52 0.44
17.78 3.97
0.34 0.03
106.8 8.6
99.4 11.8
0.35 0.03
19.26 4.02
0.37 0.09
1.5 0.38
18.17 3.60
0.33 0.03
117.5 10.4
109.2 12.6
0.37 0.03
14.53 2.95
0.22 0.04
0.89 0.17
14.81 3.09
0.37 0.03
108.3 5.4
87.7 8.1
0.37 0.03
26.24 4.15
0.39 0.06
1.68 0.24
26.95 3.86
0.39 0.03
95.0 4.5
0.35 0.03
16.66 1.84
0.08 0.01
0.51 0.06
17.26 2.01
0.34 0.03
113.0 11.5
81.2 7.7
α-Tocopherol
δ-Tocopherol
β,γ-Tocopherol
α-Tocopherol adj.
Retinol adj.
Chol (mg/dL)
TG (mg/dL)
83.5 11.1
Day 28
Retinol
0.44 0.04
19.47 4.44
0.25 0.05
1.36 0.31
18.75 4.54
0.43 0.04
110.5 6.3
104.4 13.1
0.39 0.04
22.1 4.6*
0.37 0.08a,*
1.43 0.31*
20.58 4.69*
0.37 0.04
115.8 9.5
108.2 8.2
0.41 0.03
19.84 3.88
0.28 0.05*
1.19 0.2*
20.16 3.81
0.39 0.02
116.7 5.5*
85.1 11.7
0.46 0.04
30.44 3.72*
0.43 0.05b,*
1.69 0.18a,*
30.14 3.70*
0.48 0.03
0.42 0.05
20.54 2.83*
0.12 0.03a,b
0.62 0.13a
20.73 2.99
0.40 0.05
α-Tocopherol
δ-Tocopherol
β,γ-Tocopherol
α-Tocopherol adj.
Retinol adj.
Chol (mg/dL)
TG (mg/dL)
100.0 5.4
99.7 16.0
116.6 11.0
87.4 10.2
aCommon roman letter superscripts within a row indicate a significant difference between groups; asterisk indicates a sig-
nificant change (P ≤ 0.05) from day 0 to day 28. Chol, cholesterol; TG, triglycerides; for other abbreviations see Table 1.
milk (38) and agrees reasonably well with the observed in- fants not supplemented with AA is in agreement with earlier
take of about 43 mg/kg/d in the studied breast-fed infants. reports (36,43,44). The addition of GLA either alone or in
The formulas showed a LA/α-linolenic acid ratio of about combination with fish oil did not significantly improve the
11, which was markedly different from the mean ratio of 36 in AA status as compared to the unsupplemented group. This
human milk lipids, caused by the low α-linolenic acid content confirms previous observations in studies where infant for-
of human milk. The slightly higher LA content of 15.4% in mulas had been supplemented with 0.5 or 0.3% GLA in addi-
breast milk compared to 13% in the formulas was not reflected tion to EPA and DHA (0.4 and 0.3%) or (0.6 and 0.4%), re-
by higher LA concentrations in the plasma phospholipids and is spectively, and either an unchanged or a decreased AA con-
not assumed to cause an increase in AA, as the addition of LA tent in red blood cell lipids was observed (23,24). Thus, even
to formulas showed only small effects on circulating AA (39).
in the absence of dietary n-3 LC-PUFA, an intake of up to
There were no significant differences between groups with 0.6% GLA failed to produce a significant increase of AA sta-
respect to plasma TG and total Chol concentrations, even tus. On the other hand, GLA supplementation in children and
though breast milk contains considerably more Chol than for- adults showed differing effects on AA status (18,45). John-
mulas for infants based on plant oil (40). This lack of a dif- son et al. (18) reported a significant increase of total plasma
ference might be due to the fact that the infants were studied AA percentage after 3 wk of GLA (1.5–6 g/d) intake, which
in the period when enteral feeding had just started. However, corresponded to intakes ≥20 mg/kg/d assuming a body weight
the concentrations were similar to values observed previously of 70 kg. Animal studies clearly showed that GLA is con-
in newborn infants (41,42).
verted to AA (16), but whether there is an increase of AA
Total fatty acids in plasma phospholipids did not differ be- concentrations in humans seems to depend on further factors.
tween groups or change with time and were well within the Stable isotope studies in term infants identified ∆-6 desatura-
range of previously observed values in preterm infants of this tion as the limiting step in the endogenous conversion of di-
age (36). AA levels were similar in all groups at study start, etary LA to AA (13). The principal activity of the conversion
while there were clear differences between the formula of LA via GLA and DGLA to AA in neonates has been
groups and the breast milk group at the later time points, demonstrated in vitro (46) and in vivo (12–14), but the addi-
reaching significance at day 28. The decrease with time in in- tion of the intermediate GLA to the diet does not seem to im-
Lipids, Vol. 36, no. 6 (2001)

BORAGE OIL AND PLASMA ARACHIDONIC ACID IN PREMATURE INFANTS
563
prove the yield of AA. As it can be assumed that GLA has blood cells (57). Hence, the effect of GLA supplementation
been efficiently absorbed (47), there must be some other might depend on the current AA status. As there was no AA
physiological reason.
intake in the formula groups studied and AA concentrations
Although DGLA was delivered only in trace amounts with were lower than those observed by Emken et al., in combina-
all tested formulas, DGLA showed plasma phospholipid con- tion with a possible immaturity of the desaturating enzyme sys-
centrations in the GLA-containing formula groups that were tem, these findings could explain the absence of an increase of
higher than in the HM group, but lower values were present AA after GLA supplementation.
in the ST group, although differences were not significant
This study also investigated the influence of fish oil sup-
(Table 3). It appears that 0.6% of GLA in the diet may well plementation on infant plasma phospholipid composition.
replace about 0.4% of DGLA, as provided with breast milk The addition of 0.3% DHA established phospholipid DHA
lipids (22), thus pointing toward rapid elongation. This is in values similar to those found in the HM-fed group, and even
accordance with other findings in neonates, healthy adults, exceeded them in a population with rather low HM contents
and patients with atopic dermatitis (18,24,48). A further indi- of DHA. These findings are in accordance with published
cation for an active fatty acid elongase in the studied preterm studies in term infants; e.g., Horby-Jorgensen et al. (24) re-
neonates is the finding that 20:2n-6, the elongation product of ported 1.9% DHA in red blood cell phosphatidylcholine after
LA, was found in similar concentrations in all feeding groups, feeding a formula with 0.3% DHA for 1 mon compared to
although only breast milk contained appreciable amounts of 1.25% in an unsupplemented group. In preterm infants 0.2%
this fatty acid.
DHA compared to 0% DHA in dietary fatty acids caused a
Phospholipid GLA concentrations did not respond to di- concentration difference of about 5 mg/L in plasma total
etary GLA supply, which may be explained by the low affin- phospholipid DHA after 4 wk (25). In previous studies, di-
ity of GLA toward incorporation into phospholipids (49). etary fish oil without simultaneous addition of preformed AA
Moreover, lipoprotein lipase preferentially hydrolyzes fatty into the formula had led to AA concentrations below those of
acids with 16 or 18 carbon atoms from chylomicrons as com- groups with LC-PUFA unsupplemented (25), which was ex-
pared to polyenoic fatty acids with 20 carbon atoms (50). plained by the competition of EPA and DHA for the incorpo-
There appears to be more uptake of the shorter fatty acids by ration into phospholipids (58) and an inhibitory effect of n-3
tissues for oxidation, while a lower proportion is available for fatty acids on desaturase activities (59,60). It has been postu-
conversion to LC-PUFA or direct incorporation into phospho- lated that DHA and not EPA is the potent desaturase inhibitor
lipids in the liver. This is in agreement with the observation in humans and that it mainly affects ∆-6 desaturation (61).
of Leyton et al. (51) who reported that 27% of orally admin- The absence of a further AA decrease with DHA intake may
istered GLA in rats was oxidized to CO2 within 24 h, while be explained by the GLA supply in both fish oil-supple-
only 14% each of DGLA or AA were recovered in breath. mented groups, thereby circumventing the ∆-6 desaturation
Brouwer et al. (52) and Woltil et al. (53) reported that dietary step. Effects similar to those seen here were reported by Ward
GLA is preferentially incorporated into Chol esters in adults et al. (62) in brain phospholipids of artificially reared rat pups
and that a significantly higher GLA content in plasma Chol on diets varying in their GLA content from 0.5 to 3% and
esters of low birth weight infants was found when they were with either 0.5 or 2.5% DHA (62). They demonstrated that
fed a formula with 0.3% GLA than when they were fed HM.
even 3% GLA in dietary lipids did not significantly increase
Obviously, dietary GLA was efficiently elongated to AA content compared to 0.5% GLA. In contrast to our results,
DGLA, possibly already in the intestine (54). Elongation prod- Makrides et al. (23) found a lower AA value of 10.2 1.0%
ucts and remaining precursors reached the circulation after in- (mean SD) of total fatty acids from erythrocytes in term in-
corporation into chylomicrons, and it is possible that from these fants fed for 16 wk a formula with 0.58% EPA, 0.36% DHA,
lipoproteins GLA was preferentially eliminated and oxidized. and 0.27% GLA compared to an unsupplemented control
Thus only a negligible portion of GLA might have been avail- group (12.9 1.3%), but they did not study a group fed the
able in the liver for conversion into AA and incorporation into same dose of n-3 LC-PUFA but no GLA.
phospholipids. Owing to the active elongation, the ingested
Tocopherol and retinol did not differ between groups on
GLA appears in the liver converted to DGLA, and might be day 28 in either the absolute concentrations or in α-tocoph-
handled similarly to dietary DGLA. After feeding deuterated erol and retinol concentrations after adjustment by multiple
DGLA to adults and measuring the concentration of deuterated regression. At study day 0, 50% of the infants showed α-to-
AA in plasma, it was estimated that only a very low percentage copherol concentrations below a level of 12.4 µmol/L, con-
of exogenous DGLA is converted to AA (55,56). The study by sidered as the threshold for vitamin E sufficiency (63). This
Emken et al. (56) offers a possible explanation for an inconsis- number decreased to 38% at day 28 with mean values for all
tent effect of GLA feeding on AA concentrations, as they noted groups from day 14 on above the threshold of sufficiency. Al-
that in subjects with an AA intake of 0.2 g/d, only about 2% of though there are suggestions to aim at much higher vitamin E
DGLA was desaturated, while this percentage increased to concentrations, the intake in the formula-fed groups was 1.6
18% with an AA intake of 1.7 g/d. In a larger group of subjects mg vitamin E/100 kcal (1.9 mg vitamin E/g PUFA), which is
it was demonstrated that the high AA intakes were followed by well above recommendations of the European Society for Pe-
elevated AA concentrations in all plasma lipid fractions and diatric Gastroenterology Hepatology and Nutrition (64).
Lipids, Vol. 36, no. 6 (2001)

564
H. DEMMELMAIR ET AL.
10. Koletzko, B., and Braun, M. (1991) Arachidonic Acid and Early
In conclusion, our results are in line with previous findings
Human Growth: Is There a Relation? Ann. Nutr. Metab. 35,
128–131.
11. Carlson, S.E., Cooke, R.J., Werkman, S.H., and Tolley, E.A.
(1992) First Year Growth of Preterm Infants Fed Standard Com-
pared to Marine Oil n-3 Supplemented Formula, Lipids 27,
901–907.
12. Carnielli, V.P., Wattimea, D.J.L., Luijendijk, I.H.T., Boerlage,
A., Degenhart, H.J., and Sauer, P.J.J. (1996) The Very Low
Weight Premature Infant Is Capable of Synthesizing Arachi-
donic and Docosahexaenoic Acids from Linoleic and Linolenic
Acids, Pediatr. Res. 40, 169–174.
13. Szitanyi, P., Koletzko, B., Mydlilova, A., and Demmelmair, H.
(1999) Metabolism of 13C-Labelled Linoleic Acid in New-
born Infants During the First Week of Life, Pediatr. Res. 45,
669–673.
(22,24,53,65) and show that addition of 0.6% GLA to formula
diets does not increase AA content in plasma phospholipids
of preterm infants and does not achieve values similar to com-
parable HM-fed infants. This effect does not seem to depend
on the n-3 LC-PUFA content of the diet with the dose ranges
studied. Although AA in the diet cannot be substituted by
equimolar amounts of GLA, its addition may have other ben-
eficial effects, e.g., increased high density lipoproteins (66).
For the achievement of a LC-PUFA status similar to breast-
fed infants, the supplementation of AA and n-3 LC-PUFA
seems necessary.
14. Sauerwald, T.U., Hachey, D.L., Jensen, C.L., Chen, H., Ander-
son, R.E., and Heird, W.C. (1997) Intermediates in Endogenous
Synthesis of C22:6ω-3 and C20:4ω-6 by Term and Preterm In-
fants, Pediatr. Res. 41, 183–187.
15. Gibson, R.A., and Makrides, M. (1998) The Role of Long Chain
Polyunsaturated Fatty Acids (LCPUFA) in Neonatal Nutrition,
Acta Paediatr. 87, 1017–1022.
16. Luthria, D.L., and Sprecher, H. (1995) Metabolism of
Deuterium-Labeled Linoleic, 6,9,12-Octadecatrienoic, 8,11,14-
Eicosatrienoic, and Arachidonic Acids in the Rat, J. Lipid Res.
36, 1897–1904.
ACKNOWLEDGMENTS
The authors are grateful to Dr. Oross Istváné, Tóth Lászlóné, Dr.
Kóbor Lászlóné, and Töröcsik Lászlóné for expert technical assis-
tance. This study was supported in part by Deutsche Forschungsge-
meinschaft, Bonn, Germany (Ko 912/5-2), and Hoffmann-La Roche
Ltd., Basel, Switzerland. The experimental study formulas were
kindly provided by Nutricia, Zoetermeer, The Netherlands.
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