RP-HPLC/MS-APCI analysis of branched chain TAG prepared by precursor-directed biosynthesis with rhodococcus erythropolis

Article abstract of DOI:10.1007/s11745-010-3447-7

Reversed phase liquid chromatography-atmo-spheric pressure chemical ionization mass spectrometry (RP-HPLC/MS-APCI) was used to analyze both synthetic triacylglycerols (TAG) having 1-3 branched fatty acids (FA) in the molecule, and natural TAG prepared by precursor directed biosynthesis from valine, leucine and iso-leucine and the corresponding branched short-chain acids in cultivations of Rhodococcus erythropolis. The technique made it possible to identify and quantify TAG differing in a single branched-chain FA. Altogether 11 TAG were synthesized, out of which 8 were synthesized stereospecifi-cally. Branched-and straight-chain-TAG were separated and identified while TAG differing only in iso or anteiso FA could not be separated. The APCI mass spectra of iso-, anteiso-and straight-chain TAG were completely identical. The natural material was found to contain 19 TAG having at least one branched FA. Cultivation on six different substrates showed, apart from the presumed and common incorporation of precursors to iso-even, iso-odd and anteiso FA, also some unusual features such as an increase in the content of odd-FA after the addition of Val (attributed to catabolism of Val to propionate) or the appearance of branched monounsaturated FA. The two-sample paired t test, when applied to the TAG, showed that only the pair Val and isobutyrate differ in incorporation into FA-see, e.g. proportions of M/M/O and brM/brM/O (1.2:1.2 and 1.9:1.2, respectively). Also, incorporation of Val (isobu-tyrate) yielded only TAG having two branched FA in the molecule, whereas Leu and Ile (isovalerate and 2-methyl-butyrate) gave only TAG with a single branched FA in the molecule. AOCS 2010.

Full text of DOI:10.1007/s11745-010-3447-7

Lipids (2010) 45:743–756
DOI 10.1007/s11745-010-3447-7
METHODS
RP-HPLC/MS-APCI Analysis of Branched Chain TAG Prepared
by Precursor-Directed Biosynthesis with Rhodococcus erythropolis
Olga Schreiberova´
Karel Sigler
Alena Cejkov a´ • Tom a´ sˇ Rezanka
•
Tereza Krulikovska´
•
•
ˇ
ˇ
Received: 26 May 2010 / Accepted: 29 June 2010 / Published online: 16 July 2010
Ó AOCS 2010
Abstract Reversed phase liquid chromatography–atmo-
spheric pressure chemical ionization mass spectrometry
1.9:1.2, respectively). Also, incorporation of Val (isobu-
tyrate) yielded only TAG having two branched FA in the
molecule, whereas Leu and Ile (isovalerate and 2-methyl-
butyrate) gave only TAG with a single branched FA in the
molecule.
(
RP-HPLC/MS-APCI) was used to analyze both synthetic
triacylglycerols (TAG) having 1–3 branched fatty acids
FA) in the molecule, and natural TAG prepared by pre-
(
cursor directed biosynthesis from valine, leucine and iso-
leucine and the corresponding branched short-chain acids
in cultivations of Rhodococcus erythropolis. The technique
made it possible to identify and quantify TAG differing in a
single branched-chain FA. Altogether 11 TAG were syn-
thesized, out of which 8 were synthesized stereospecifi-
cally. Branched- and straight-chain-TAG were separated
and identified while TAG differing only in iso or anteiso
FA could not be separated. The APCI mass spectra of iso-,
anteiso- and straight-chain TAG were completely identical.
The natural material was found to contain 19 TAG having
at least one branched FA. Cultivation on six different
substrates showed, apart from the presumed and common
incorporation of precursors to iso-even, iso-odd and anteiso
FA, also some unusual features such as an increase in the
content of odd-FA after the addition of Val (attributed to
catabolism of Val to propionate) or the appearance of
branched monounsaturated FA. The two-sample paired
t test, when applied to the TAG, showed that only the pair
Val and isobutyrate differ in incorporation into FA—see,
e.g. proportions of M/M/O and brM/brM/O (1.2:1.2 and
Keywords Rhodococcus erythropolis ꢀ
RP-HPLC/MS-APCI ꢀ Branched chain triacylglycerols ꢀ
Precursor directed biosynthesis
Abbreviations
GC-MS
Gas chromatography–mass
spectrometry.
LC-MS
Liquid chromatography–mass
spectrometry
RP-HPLC/MS-APCI Reversed phase liquid
chromatography–atmospheric
pressure chemical ionization mass
spectrometry
FA
Fatty acid(s)
TAG
DAG
MAG
BrFA
BrTAG
i-Bu
Triacylglycerol(s)
Diacylglycerol(s)
Monoacylglycerol(s)
Branched (i or ai) FA
Branched (i or ai) TAG
Isobutyric acid
i-Va
Isovaleric acid
ˇ
O. Schreiberov a´ ꢀ T. Krulikovsk a´ ꢀ A. Cejkov a´
2-MeBu
2-Methylbutyric acid
Valine
Department of Fermentation Chemistry and Bioengineering,
Institute of Chemical Technology Prague, Technick a´ 5,
Val
1
66 28 Prague, Czech Republic
Leu
Leucine
Ile
Isoleucine
ˇ
K. Sigler ꢀ T. Rezanka (&)
DMAP
DCC
ACN
4-Dimethylaminopyridine
0
Institute of Microbiology, Academy of Sciences of the Czech
Republic, V ´ı de nˇ sk a´ 1083, 142 20 Prague, Czech Republic
e-mail: rezanka@biomed.cas.cz
N,N -Dicyclohexylcarbodiimide
Acetonitrile
1
23

7
44
Lipids (2010) 45:743–756
with the given branched-FA depending on the starter unit
iPrOH
2-Propanol
(
addition of Val produces even-iso-FA, addition of Leu
odd-iso-FA, addition of Ile odd-ai-FA). This fact has been
noted many times in myxobacteria [9], in Nonomuraea sp.
i-14:0 (iM)
12-Methyltridecanoic acid
Tetradecanoic acid
1
4:0 (M)
i-15:0 (iX)
13-Methyltetradecanoic acid
12-Methyltetradecanoic acid
Pentadecanoic acid
[10], Propionibacterium freudenreichii [11], Staphylococ-
cus xylosus [12] or S. carnosus [13].
ai-15:0 (aiX)
1
5:0 (X)
i-16:0 (iP)
6:0 (P)
i-16:1 (iPo)
6:1 (Po)
Current studies have shown that branched chain FA,
predominantly i-15:0 (iso-pentadecanoic) and ai-15:0
14-Methylpentadecanoic acid
Hexadecanoic acid
1
(
anteiso-pentadecanoic) inhibit the growth of various can-
14-Methylpentadecenoic acid
Hexadecenoic acid
cer cell lines both in vitro and in vivo [14, 15]. Other
experiments have documented that their activity decreases
with an increase or decrease in chain-length from i-16:0
1
i-17:0 (iMa)
15-Methylhexadecanoic acid
14-Methylhexadecanoic acid
Heptadecanoic acid
ai-17:0 (aiMa)
(
isopalmitic) [16]. This effect is explained as being due to
1
7:0 (Ma)
cancer cells being more dependent on fatty acid biosyn-
thesis than healthy cells.
i-17:1 (iMo)
15-Methylhexadecenoic acid
14-Methylhexadecenoic acid
Heptadecenoic acid
ai-17:1 (aiMo)
As shown in our earlier study of Rhodococcus lipids [17],
LC–MS analysis of odd-chain TAG is still largely unex-
plored, mostly due to the scarcity of sources with odd-chain
TAG. The situation with branched-chain TAG from natural
sources is even worse. To our knowledge only two sources of
branched TAG have so far been identified, both of them of
bacterial origin. Metz et al. [18] used GC–MS to separate and
identify intact TAG from Streptomyces avermitilis contain-
ing odd- and even-chain FA including iso- and anteiso-FA.
Another source of branched TAG is milk; branched FA
contained in it are largely derived from bacteria leaving the
rumen [14, 15]. With one exception [19], even here branched
TAG have not been separated from straight-chain TAG. All
other studies [20, 21] mention only odd-chainTAG, although
branched-TAG also have to be present, as seen from the
analysis of FA in butter [19, 21]. Figure 1 in the paper by
Marai et al. [21] clearly shows the presence, between peaks
1
7:1 (Mo)
i-18:0 (iS)
8:0 (S)
i-18:1 (iO)
8:1 (O)
16-Methyloctadecanoic acid
Octadecanoic acid
1
16-Methyloctadecenoic acid
Octadecenoic acid
1
Introduction
Many prokaryotic microorganisms such as Mycobacterium
sp., Nocardia sp., Micromonospora sp., Dietzia sp., Gor-
donia sp., Streptomyces sp. and Rhodococcus sp. accu-
mulate large amounts of lipids in lipid bodies in the cells
and mycelia [1, 2]. In the genus Rhodococcus, the infor-
mation about the structure of lipids in the cytoplasmic lipid
bodies, in particular triacylglycerols (TAG), is rather
scarce [3, 4].
4
8:0 (PPP) and 49:0 (PPMa), of two unidentified peaks that
could belong to TAG 49:0 having branched margaric acids
i.e. i-17:0 and/or ai-17:0). Only Myher et al. [19] have given
As a carbon source, the genus Rhodococcus can utilize
very unusual compounds such as hexadecane [5] or phen-
yldecane that gives rise to triacylglycerol in which one
fatty acid was replaced by a phenyldecanoic acid residue
(
the TCN (theoretical carbon number) for branched TAG 0.38
less than for straight-chain TAG; to our knowledge this is
thus the only paper that has explored the LC–MS of branched
TAG.
[
6], or 1-chloro-, l-bromo- and 1-iodohexadecane that
ultimately yield x-halogen fatty acids (FA) [7]. Further-
more, alkanes can be directly oxidized and activated to the
respective acyl-CoA thioesters and incorporated into TAG
Based on our previous experience with the cultivation of
Rhodococcus [22] and with LC–MS of lipids [23, 24] we
used here precursor directed biosynthesis to prepare bran-
ched TAG, which were qualitatively and quantitatively
analyzed by using synthetic standards.
[
3, 8]. The composition of the TAG is known to vary
strongly when the cells are cultivated on propionic acid,
which is activated to propionyl-CoA, resulting in a much
higher fraction of odd-numbered FA (over 95% total FA)
[
8]. With valerate addition the proportions of odd-chain FA
Experimental
can reach nearly 85% total FA.
With other bacteria, the addition of branched amino
acids, i.e. Val, Leu or Ile, or their metabolites such as
isobutyric (i-Bu), isovaleric (i-Va) or 2-methylbutyric
Microbial Material
Rhodococcus erythropolis CCM 2595 obtained from the
Czech Collection of Microorganisms (Masaryk University,
Brno, Czech Republic) was subjected to a 6-month
(
2-MeBu) acids into the culture medium induces the pro-
duction of increased amounts of branched FA enriched
1
23

Lipids (2010) 45:743–756
745
1
00
carbodiimide (DCC), and standards of glyceryl tripalmitate
and glyceryl triheptadecanoate were purchased from Sigma-
Aldrich (Prague, CR). The mono- and diacylglycerols were
purchased from Larodan Fine Chemicals, Malmo, Sweden.
9
8
7
6
5
4
3
2
1
0
0
0
0
0
0
0
0
0
0
Preparation of Mixed FA Composition TAG
The esterification of glycerol with acids has been described
previously [25]. Briefly, a rapidly stirred suspension of
1
,3-diacylglycerol or 1,2-diacylglycerol (10 lmol), the acid
11 lmol), and a catalytic amount of 4-dimethylamino-
pyridine (2 lmol) in anhydrous CH Cl (5 mL) was treated
(
2
2
0
with N,N -dicyclohexyl-carbodiimide (12 lmol). The
resulting mixture was stirred under N for 24 h at room
2
temperature, diluted with CH Cl , and filtered to remove
2
2
precipitated 1,3-dicyclohexyl urea. The filtrate was washed
with 0.5 N HCl, saturated aqueous NaHCO , H O, and
3
2
brine and dried (MgSO ). The solvent was removed in
4
vacuo, and the remaining residue was purified by TLC
(
(
silica gel H, developed in hexane/diethyl ether/acetic acid
70:30:1, by vol.). The yields of appropriate TAG are in
Table 1. The purity (see also Table 1) of individual syn-
thesized TAG was determined by RP-HPLC, for the con-
ditions see below.
670
680
690
700
710
720
730
Preparation of Single FA Composition TAG
time (min)
Fig. 1 RP-HPLC/APCI-MS chromatogram of three synthetic TAG
Ma/Ma/Ma, iMa/iMa/iMa, and aiMa/aiMa/aiMa)
The TAG were prepared as described above, only the ratios
(
of FA and glycerol were different, i.e. for 10 lmol of FA,
3
lmol of glycerol, 4 lmol of 4-dimethylaminopyridine,
0
physiological adaptation to phenol. The strain reached a high
growth rate in the presence of succinate (10 g/L) used as the sole
carbon source. Cells were grown in the basic mineral medium
and 36 lmol of N,N -dicyclohexyl-carbodiimide was used.
Further separation and purification was identical, see
above. The yields are in Table 1.
(
g/L: KH PO 0.17; MnCl ꢀ4H O 0.001; K HPO 0.13;
The lyophilized cells were mixed with 10 mL of hexane
and the mixture was stirred for 15 min. The cells were
filtered off, hexane was evaporated and the oil samples
2
4
2
2
2
4
CaCl ꢀ2H O 0.00026; (NH ) SO 0.71; FeSO ꢀ7H O 0.0006;
2
2
4 2
4
4
2
MgCl ꢀ6H O 0.34; Na MoO ꢀ2H O 0.002; pH was adjusted to
2
2
2
4
2
7
.0) containing alternatively amino acids or short carboxylic
acids (sodium salts) as the carbon source (valine, leucine,
isoleucine, isovalerate, isobutyrate, 2-methyl butyrate—each
Table 1 Synthesis of TAG
3
g/L) and 1 g/L of succinate as cosubstrate. The cultivations
Alcohol (1 lmol)
Acid/lmol TAG
Yield Purity
(
%)
(%)
were performed on a rotary shaker at 100 rpm and 30 °C.
The cells were collected at the end of exponential phase
Glycerol
Glycerol
Glycerol
Ma/3.15
iMa/3.15
Ma/Ma/Ma
iMa/iMa/iMa
92
90
C99
C99
C99
C99
C99
C99
[98
[98
[98
[98
[97
(
97 h) by centrifugation (10 min, 9,050g), washed twice
with physiological solution and lyophilized (dry biomass
yield depending on the C-sources: 353 mg/L—valine,
aiMa/3.15 aiMa/aiMa/aiMa 87
1
1
1
1
1
1
1
1
-Monopalmitin
-Monopalmitin
-Monopalmitin
,2-Dipalmitin
,2-Dipalmitin
,2-Dipalmitin
,3-Dipalmitin
Ma/2.10
iMa/2.10
P/Ma/Ma
75
73
76
69
68
70
68
64
4
38 mg/L—leucine, 663 mg/L—isoleucine, 690 mg/L—
P/iMa/iMa
isovalerate, 583 mg/L—isobutyrate, 645 mg/L—2-methyl
butyrate, 890 mg/L—succinate).
aiMa/2.10 P/aiMa/aiMa
Ma/1.05
iMa/1.05
P/P/Ma
P/P/iMa
Standards and Isolation
aiMa/1.05 P/P/aiMa
Ma/1.05
P/Ma/P
Acetonitrile, 2-propanol, hexane, dichloromethane, gly-
0
,3-Diheptadecanoin P/1.05
Ma/P/Ma
cerol, 4-dimethylaminopyridine (DMAP), N,N -dicyclohexyl-
1
23

7
46
Lipids (2010) 45:743–756
were dissolved in an acetonitrile-2-propanol-hexane mix-
ture (1:1:1, v/v/v), which was injected on the column.
mixture (99:1, v/v) to concentrations of 1, 5, 10, 50, and
100 lg/mL. All calibration curves were measured using a
10 lL injection volume of solutions in three repeated
FAME Analysis
analyses and the average peak areas were used for the
construction of calibration curves. For reliable quantitation,
concentrations of individual TAG in analyzed samples
have to be in a given interval (after a suitable dilution). The
limits of detection at S/N = 3 were determined with the
injection volume 10 lL and averaged as 1 lg/mL for sat-
urated TAG. RFs are expressed relative to Ma/Ma/Ma,
which is set to 1.00. For MS-APCI a and b values are
coefficients of the linear calibration dependence y =
The TAG (10 mg) were saponified overnight in 10%
KOH–MeOH at room temperature. A fatty acid fraction
obtained from saponification was partitioned between
alkali solution (pH 9) and Et O to remove basic and neutral
2
components. The aqueous phase, containing FA, was
acidified to pH 2 and extracted with hexane. The fatty acid
fraction was methylated using CH N . GC–MS of fatty
2
2
acid methyl ester (FAME) mixture was done on a Finnigan
020 B in EI mode. Splitless injection was at 100 °C, and a
fused silica capillary column (Supelcowax 10; 60 m 9
.25 mm i.d., 0.25 mm film thickness; Supelco, Prague)
ax ? b and RFs are calculated as RF = aMa/Ma/Ma/aTAG,
2
1
because b values can be neglected. Here r is the value of
coefficient of determination, y corresponds to the peak
areas and x is the concentration in lg/mL.
0
was used. The temperature program was as follows: 100 °C
for 1 min, subsequently increasing at 20 °C/min to 180 °C
and at 2 °C/min to 280 °C, which was maintained for
Data acquisition and analyses were performed using PC
with MassLab 2.0 for Windows XP applications/operating
software.
1
min. The carrier gas was helium at a linear velocity of
6
0 cm/s. All spectra were scanned within the range of
m/z 50–500. The structures of FAME were confirmed by
comparison of retention times and fragmentation patterns
with those of the standard FAME (Supelco, Prague).
Results and Discussion
As seen in Table 2, FA from Rhodococcus comprise not
only odd-chain FA [17], but also branched-chain FA that
RP-HPLC/MS-APCI
The HPLC equipment consisted of a 1090 Win system,
PV5 ternary pump and automatic injector (HP 1090 series,
Agilent, USA) and two Hichrom columns HIRPB-250AM
Table 2 Fatty acid compositions (%) of seven cultivations of R.
erythropolis at 30 °C with different carbon sources [succinate, Val,
Leu, Ile, isobutyric (i-Bu), isovaleric (i-Va) and 2-methyl-butyric
(
2-MeBu) acid] as determined by GC–MS
2
50 9 2.1 mm ID, 5-lm particle size, in series. This setup
provided us with a high-efficiency column—approximately
6,000 plates/250 mm. A quadrupole mass spectrometer
Succinate Val i-Bu Leu i-Va Ile
2-MeBu
–
2
a
i-14:0 (iM)
–
8.1 6.7
–
–
–
system Navigator (Finnigan MAT, San Jose, CA, USA)
was used for analysis. The instrument was fitted with an
atmospheric pressure chemical ionization source [vaporizer
temperature 390 °C, capillary heater temperature 260 °C,
corona current 7 lA, sheath gas—high-purity nitrogen,
pressure 0.45 MPa, and auxiliary gas (also nitrogen)
flow rate 15 mL/min]. Positively charged ions with
m/z 200–1,000 were scanned with a scan time of 0.5 s. The
whole HPLC flow (0.35 mL/min) was introduced into the
APCI source without any splitting. TAG were separated
using a gradient solvent program with acetonitrile (ACN)
and 2-propanol (iPrOH) as follows: initial ACN/iPrOH
1
4:0 (M)
9.8
–
6.3 5.9 4.5 5.3 3.8 4.4
i-15:0 (iX)
–
–
–
–
14.8 14.2
–
–
ai-15:0 (aiX)
–
–
–
19.1 20.4
1
5:0 (X)
i-16:0 (iP)
6:0 (P)
i-16:1 (iPo)
6:1 (Po)
8.2
–
13.5 14.2 11.6 10.8 7.6 6.7
18.2 17.6
12.1 13.0 16.3 15.7 11.2 10.8
0.3 0.2
13.1 14.1 13.1 14.5 10.9 11.3
–
–
–
–
1
19.3
–
–
–
–
1
18.5
–
i-17:0 (iMa)
–
–
–
–
19.5 20.2
–
–
ai-17:0 (aiMa)
–
–
–
21.3 19.7
1
7:0 (Ma)
4.9
–
9.3 8.8 3.4 4.1 4.2 4.6
i-17:1 (iMo)
–
–
–
–
0.7 0.6
–
–
(
99:1, v/v); linear from 5 to 120 min ACN/iPrOH 30:70,
ai-17:1 (aiMo)
–
–
–
0.6 0.5
v/v); held until 30 min; the composition was returned to the
initial conditions over 10 min. Isocratic separation by the
mixture ACN/iPrOH 65:35, v/v) was used for resolution of
the regioisomers. A peak threshold of 0.07% intensity was
applied to the mass spectra.
1
7:1 (Mo)
i-18:0 (iS)
8:0 (S)
i-18:1 (iO)
9.2
–
6.7 7.1 5.4 4.8 5.7 6.2
0.6 0.5
1.8 1.6 0.9 0.6 2.8 2.9
0.4 0.5
9.6 9.8 9.8 9.2 12.8 12.5
–
–
–
–
1
3.1
–
–
–
–
–
1
8:1 (O)
27.0
The saturated synthesized TAG (Ma/Ma/Ma, iMa/iMa/
iMa, and aiMa/aiMa/aiMa) were dissolved in ACN/iPrOH
a
Minority fatty acids up to 0.1% of total fatty acids were omitted
1
23

Lipids (2010) 45:743–756
747
were produced by precursor directed biosynthesis from
branched chain precursors. In view of the scarcity of data
on branched chain TAG (see above) we decided to syn-
thesize a series of standards (see Table 1) that were used to
verify the basic chromatographic and mass spectral char-
acteristics. The standards were synthesized by conventional
procedures, as simply as possible and with the highest
yield. We followed the procedure described by Lie Ken Jie
and Lam [26] who synthesized TAG of AAB, ABA, BBA
and BAB types. Commercially available glycerol, mono-
acyl- and diacylglycerols (1-palmitin, 1,2-dipalmitin,
100
P/Ma/Ma
P/aiMa/aiMa
90
80
70
60
50
40
30
20
10
0
1
,3-dipalmitin and 1,3-diheptadecanoin) yielded a total
of 11 TAG by mild organic synthesis performed at 25 °C
for 24 h under catalysis with dicyclohexylcarbodiim-
ide (DCC) and 4-dimethylaminopyridine (DMAP) (see
‘‘Experimental’’).
Individual TAG were analyzed by RP-HPLC. Figure 1
shows the chromatographic analysis of three synthetic
TAG, Ma/Ma/Ma, iMa/iMa/iMa, and aiMa/aiMa/aiMa.
The separation of straight-chain TAG from both branched-
chain TAG presented no problems while separation of iMa/
iMa/iMa from aiMa/aiMa/aiMa was hours-long—a time
span unacceptable in the analysis of a natural sample—and
succeeded only under isocratic conditions using the mobile
phase ACN-iPrOH (65:35, v/v).
Chromatographic separation of branched TAG having
two branched FA in the molecule (two i-17:0 or two
ai-17:0) from straight-chain counterparts presents no prob-
lems—gradient elution provided a base-line separation of
P/Ma/Ma from P/aiMa/aiMa or P/Ma/Ma from P/iMa/iMa,
as shown in Figs. 2 and 3. Separation of two branched
TAG such as P/iMa/iMa and P/aiMa/aiMa could be
accomplished only by isocratic elution and only with
synthetic TAG. We ascribe this phenomenon to the much
higher complexity of the natural mixture that results in a
mutual influencing and overlapping of peaks.
6
70
680
690
700
time (min)
710
720
730
Fig. 2 RP-HPLC/APCI-MS chromatogram of two synthetic TAG (P/
Ma/Ma and P/aiMa/aiMa)
?
i.e. [aiMa/aiMa] ) at m/z 579 arising by the loss of
?
margarate [RCOO?58] called also ([MAG] , i.e. [aiMa] )
?
?
?
?
?
at m/z 327, and [RCO] ion (FA , i.e. aiMa ) at m/z 253,
see also Table 3 and Fig. 4.
APCI mass spectra of all synthetic TAG, i.e. Ma/Ma/
Ma, iMa/iMa/iMa, and aiMa/aiMa/aiMa were completely
identical. Minute differences in the intensity of ions at
m/z 41, 43, 55, and 57 can be found only in electron
impact mass spectra. These ions arise probably by
splitting of the termini of aliphatic chains although Dinh-
Nguyen [28] states, based on the comparison with deu-
terium-labeled compounds, that the situation is more
complex. Unfortunately, the HPLC-electron impact con-
nection is not feasible and the determination of all three
chain types, i.e. n-, iso- and anteiso- would always
require a comparison of natural TAG with a synthetic
TAG standard.
In the case of TAG having a single branched FA we
succeeded in separating only those TAG that differed in the
site of branching. The separation was successful only in the
pairs P/P/Ma and P/P/iMa, and P/P/Ma and P/P/aiMa, in
both the synthetic and the natural mixture and with both
isocratic and gradient elution. The separation P/P/iMa and
P/P/aiMa was unsuccessful.
Positional isomers such as P/P/Ma and P/Ma/P or P/Ma/
Ma and Ma/P/Ma were partially separated only with iso-
cratic elution (see [27] for positional isomers, i.e. POP and
PPO). These two pairs were much more poorly separated
than positional isomers in which individual FA differ in
two methylene groups, such as P/P/S and P/S/P [17].
The APCI mass spectra of an AAA type TAG are very
simple. For instance, trianteisomargarin (aiMa/aiMa/aiMa),
The above data clearly show that any combination of
branched- and straight-chain acyls in TAG cannot be
resolved based on mass spectrum. Even so we performed
another experiment with synthetic TAG (P/Ma/Ma and
P/aiMa/aiMa, see Figs. 5, 6) that could facilitate the
?
?
exhibits four clusters of ions, i.e. [M?H] ([TAG] , i.e.
?
aiMa/aiMa/aiMa] ) at m/z 849, [M-RCOO] ion ([DAG] ,
?
?
[
1
23

7
48
Lipids (2010) 45:743–756
1
00
resolution of individual acyls by using MS/MS. However,
?
the MS/MS of the daughter ion at m/z 253 ([RCO] ), i.e.
P/Ma/Ma
P/iMa/iMa
ion with the same m/z value for all three acyls of all three
acids (margaric, iso-margaric and anteiso-margaric acids)
failed to reveal any perceptible change in the intensity of
ions in the interval of 35–253 Da (spectrum not shown);
this result finally excluded the possibility of distinguishing
branched- and straight-chain TAG by MS-APCI.
9
0
0
0
0
0
0
0
0
0
0
8
7
6
5
4
3
2
1
The mass spectrum of ABB type TAG such as P/aiMa/
aiMa (Fig. 6), which contains two FA, exhibits, in addition
?
to [M?H] at m/z 835, also two acylium ions, with m/z at
?
?
39 (P ) and m/z 253 (aiMa ), respectively. The spectrum
2
?
naturally features also ions of the type [MAG] at m/z 313
?
[P] ) and m/z 327 ([aiMa] ), respectively. Like with
?
(
?
aiMa/aiMa/aiMa, also [DAG] ions caused by the loss of
margarate at m/z 565 and palmitate at m/z 579 are present,
?
?
resulting in ([P/aiMa] (type [AB] ) and [aiMa/aiMa]
?
?
type [BB] ) ions, respectively.
(
Similarly, the mass spectrum of an ABC type TAG, i.e.
TAG containing three different FA, such as P/brMa/S, see
?
Fig. 7, shows three triplets of ions—three [M-RCOO] ions
at m/z 565, 579, and 593, corresponding to loss of stearate to
?
give [brMa/P] , loss of margarate to give [P/S] , and pal-
?
?
mitate to give [brMa/S] , respectively. Another triplet,
?
?
MAG] at m/z 341, 327, and 313 is formed by [S] ,
[
[
(
?
brMa] , and [P] ions. The last triplet belongs to ions S
?
?
6
70
680
690
700
710
720
730
?
?
m/z 267), brMa (m/z 253), and P (m/z 239).
Some authors [17, 29–32] observed that the relative
time (min)
?
?
Fig. 3 RP-HPLC/APCI-MS chromatogram of two synthetic TAG
P/Ma/Ma and P/iMa/iMa)
intensities of the [M-RCOO] ions, i.e. [DAG] , could be
used to obtain information on the positions of FA within
(
Table 3 TAG identified in R. erythropolis, their retention times (RT), acyl carbon number (ACN), the masses of protonated molecules, and
characteristic fragment ions of TAG
e
?
?
?
?
Peak no.
TAG
RT
ACN:n
[M?H]
[DAG]
Po/O
m/z
[DAG]
m/z
[DAG]
m/z
1
2
3
4
5
6
7
8
9
Po/Po/O
brX/Po/Po
Po/Po/Mo
X/Po/Po
M/Po/O
50.7
51.0
51.2
51.6
53.4
53.5
53.8
54.3
54.7
55.1
56.4
58.2
58.4
60.9
61.3
61.9
62.6
62.9
63.1
50:3
47:2
49:3
47:2
48:2
46:1
48:2
46:1
46:1
44:0
44:0
51:3
49:2
52:3
50:2
50:2
50:2
48:1
46:0
829
789
815
789
803
777
803
777
777
751
751
843
817
857
831
831
831
805
779
575
535
561
535
575
549
549
549
549
523
523
589
521
603
603
575
577
549
551
Po/Po
Po/Po
Po/Po
Po/Po
M/Po
547
547
547
547
521
495
547
523
495
495
495
561
561
575
549
549
547
551
523
–
–
brX/Po
Po/Mo
X/Po
Po/O
brM/O
P/Po
–
–
–
–
–
–
M/O
549
–
brM/brM/O
P/Po/Po
brM/brM
Po/Po
M/P
–
–
–
M/P/Po
P/Po
M/Po
521
–
M/M/O
M/O
M/M
–
1
1
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
8
9
brM/brM/P
M/M/P
brM/P
M/P
brM/brM
M/M
–
–
–
–
Po/Mo/O
P/Po/Mo
Po/O/O
Mo/O
Mo/P
O/O
Po/Mo
Po/Mo
Po/O
Po/O
575
549
–
Po/P
–
M/O/O
O/O
M/O
–
–
P/Po/O
Po/O
Po/S
P/Po
P/O
–
577
–
Po/Po/S
Po/Po
brP/brP
M/brP
brP/brP/Po
M/brP/brP
brP/Po
brP/brP
–
–
–
–
1
23

Lipids (2010) 45:743–756
749
Table 3 continued
e
?
?
?
?
Peak no.
TAG
RT
ACN:n
[M?H]
[DAG]
m/z
[DAG]
M/P
m/z
[DAG]
m/z
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
5
6
6
6
6
6
6
6
6
6
6
7
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
8
9
0
1
2
3
4
5
6
7
8
9
0
M/P/O
63.5
63.9
64.2
64.7
65.1
65.2
65.5
65.6
65.9
66.2
66.3
66.7
67.1
69.0
69.5
70.8
70.9
72.2
72.6
73.1
74.5
74.7
74.9
75.3
75.6
76.2
76.3
76.7
77.1
77.2
77.2
78.1
78.2
79.4
81.4
84.2
85.5
86.4
85.6
86.6
86.7
86.8
87.0
87.8
88.0
88.2
88.3
88.9
89.6
93.6
96.2
97.1
48:1
46:0
48:1
46:0
52:3
46:0
48:1
49:1
53:3
51:2
49:1
51:2
51:2
54:3
52:2
52:2
50:1
50:1
50:1
50:1
53:2
51:1
48:0
53:2
51:1
48:0
51:1
48:0
51:1
51:1
49:0
49:0
52:1
54:2
52:1
50:0
50:0
51:0
50:0
51:0
50:0
50:0
53:1
53:1
51:0
51:0
53:1
51:0
54:1
52:0
53:0
53:0
805
779
805
779
857
779
805
819
871
845
819
845
845
885
859
859
833
833
833
833
873
847
807
873
847
807
847
807
805
847
821
821
861
887
861
835
835
849
835
849
835
835
875
875
849
849
875
849
889
863
877
877
P/O
577
551
549
551
589
551
577
603
603
577
603
589
577
603
603
605
577
577
579
605
603
605
551
603
605
551
577
579
591
577
565
565
607
605
605
579
579
579
579
579
607
579
605
605
565
579
607
565
605
579
607
607
523
495
551
523
575
495
521
563
589
537
563
521
537
603
577
577
551
551
549
549
591
563
551
591
563
–
M/O
549
–
brM/brM/S
P/P/Po
brM/S
P/Po
P/P
brM/brM
P/P
–
–
–
M/P/P
M/P
–
–
Mo/Mo/O
M/M/S
Mo/O
M/S
Po/S
O/O
O/O
P/O
Mo/Mo
M/M
M/Po
brX/O
Mo/O
brX/P
X/O
–
–
–
–
M/Po/S
brX/O/O
MoOO
M/S
–
551
–
–
–
brX/P/O
X/O/O
brX/O
–
563
–
O/O
Mo/O
P/O
P/Mo/O
X/P/O
P/Mo
X/P
P/O
X/O
–
577
563
–
O/O/O
O/O
O/O
S/O
–
P/O/O
P/O
–
Po/S/O
Po/S
brP/brP
P/P
Po/O
–
575
–
brP/brP/O
P/P/O
brP/O
P/O
–
–
Po/P/S
P/S
Po/P
M/O
brMa/O
brX/O
brP/P
Ma/O
X/O
Po/S
M/S
–
577
551
–
M/O/S
O/S
brMa/O/O
brX/O/S
brP/brP/P
Ma/O/O
X/O/S
O/O
O/S
brX/S
–
565
–
brP/brP
O/O
–
–
O/S
X/S
–
565
–
P/P/P
P/P
–
brMa/O/P
M/P/S
O/P
brMa/O
M/P
591
523
521
591
551
551
577
603
577
551
551
–
brMa/P
565
551
579
565
–
P/S
M/S
P/Mo/S
Ma/O/P
P/P/brMa
P/P/Ma
Po/S/S
Mo/S
O/P
P/Mo
Ma/O
P/P
P/S
Ma/P
brMa/P
Ma/P
S/S
–
P/P
–
–
Po/S
O/O
–
–
O/O/S
O/S
–
–
P/O/S
O/S
P/O
P/S
579
–
brP/brP/S
P/P/S
brP/S
P/S
brP/brP
P/P
–
–
–
iMa/iMa/iMa
P/brMa/brMa
aiMa/aiMa/aiMa
M/S/S
iMa/iMa
brMa/brMa
aiMa/aiMa
S/S
–
–
–
brMa/P
–
565
–
–
–
–
–
M/S
551
565
593
593
593
–
–
–
Ma/Ma/P
brMa/S/O
Ma/S/O
P/brMa/S
Ma/Ma/Ma
Mo/S/S
P/Ma/S
O/S/S
Ma/Ma
S/O
Ma/P
brMa/S
Ma/S
brMa/S
–
–
–
brMa/O
591
591
579
–
S/O
Ma/O
brMa/P
Ma/Ma
S/S
P/S
–
Mo/S
Ma/S
S/S
591
593
607
607
593
593
–
–
Ma/P
S/O
P/S
–
579
–
P/S/S
S/P
S/S
–
–
brMa/S/S
Ma/S/S
S/S
brMa/S
Ma/S
–
–
S/S
–
–
e
ACN acyl carbon number, n number of double bond(s), RT experimental retention time (min)
1
23

7
50
Lipids (2010) 45:743–756
Fig. 4 Mass spectrum (APCI)
of aiMa/aiMa/aiMa
Fig. 5 Mass spectrum (APCI)
of molecular species of TAG
(
i.e. P/Ma/Ma)
Fig. 6 Mass spectrum (APCI)
of molecular species of TAG
(
i.e. P/aiMa/aiMa)
1
23

Lipids (2010) 45:743–756
751
Fig. 7 Mass spectrum (APCI)
of P/brMa/S, i.e. molecular
species isolated from
Rhodococcus
Fig. 8 Mass spectrum (APCI)
of molecular species
(
i.e. P/Ma/P)
?
The [P/P] :[P/Ma] ratio observed for P/Ma/P (Fig. 8)
?
the TAG. A rule was formulated about the energetically
less favorable loss of FA from position sn-2 and formation
?
is only 35:100, indicating that the formation of the 1,3-
?
[PP] ion is energetically unfavorable. On the other hand,
?
of [M-RCOO] i.e. 1,3-[DAG] ions. The cleavage of
these bifunctional TAG gives rise to an isomeric pair of the
?
?
in P/P/iMa (Fig. 9) the [P/P] :[P/iMa] ratio is 80:100 and
the rule can thus be used to determine the structure of this
positional isomer. This is in agreement with the result of
the organic synthesis. Apart from determining the structure
of natural compounds the rule can be used for confirming
the structure of synthesized standards, since the synthesis
may involve racemization or the original diacylglycerols
need not contain only pure sn-1,2 or sn-1,3 positional
isomer. Consequently, the two regioisomers show two
different spectra.
?
?
same [DAG] ions, i.e. [AA] and [AB] . The ratio of
?
?
AA] :[AB] was found to be lower for the ABA isomer,
?
[
?
since formation of the 1,2-isomer of the [AB] ion is
energetically more favorable than generating the analogous
?
,3-[AB] ion from the AAB isomer. If these two frag-
1
ments were energetically equivalent, then the intensity of
?
?
ions [AA] and [AB] should be in a 1:2 ratio irrespective
of the position of the A or B acyl substitution on the
glycerol backbone, which does not occur—see, e.g. Figs. 8
and 9. The positional isomers understandably differed in
To show that even a complex biological material such as
R. erythropolis biomass can yield mass spectra that make it
possible to identify TAG, we present in Figs. 10 and 11 the
spectra of brM/brM/O and also brX/P/O. Identification of
?
?
the intensity of ions types [P/P] and [P/iMa] since the
?
least abundant [M-RCOO] ion is known to correspond to
loss of the FA from the sn-2 position.
1
23

7
52
Lipids (2010) 45:743–756
Fig. 9 Mass spectrum (APCI)
of molecular species (i.e. P/P/
iMa)
Fig. 10 Mass spectrum (APCI)
of brM/brM/O, i.e. molecular
species isolated from
Rhodococcus
Fig. 11 Mass spectrum (APCI)
of brX/P/O, i.e. molecular
species isolated from
Rhodococcus
1
23

Lipids (2010) 45:743–756
753
these and other TAG was performed as described above
and given in Table 3.
The presence of Ile and 2-MeBu in the medium caused
an expected increase [11] in ai-FA (ai-15:0 to 19.1% with
Ile and 20.4% with 2-MeBu; ai-17:0 to 21.3% with Ile and
19.7% with 2-MeBu). These C-sources had the greatest
effect on the FA composition, the content of ai-FA
increasing to 40% of total FA.
As expected, and in agreement with literature data,
addition of Val and isobutyrate [10, 32] induced the bio-
synthesis of branched FA. No i- or ai-FA, neither odd- nor
even-chain ones, were detected in control cultivation
whereas the addition of any of the two precursors caused an
increase of i-14:0 from 0 to 8.1% and a rise of i-16:0 from
Following the addition of given precursors, correspond-
ing branched-chain monoenoic FA were identified in
amounts of about 0.5% total FA. In contrast to straight-chain
monoenoic FA only C17 and C18 branched-chain monoe-
noic FA were identified. This phenomenon can be explained
in two ways. The first possibility is that the appropriate
desaturase has a higher affinity for longer chains; this cor-
responds with the fact that no straight-chain monoenoic C14
and C15 FA were found. The other possibility is that the high
melting point of straight-chain FA lowers the membrane
fluidity to an extent that forces the bacterium to biosynthe-
size branched-chain monoenoic FA with a lower melting
point in order to enhance membrane fluidity. The melting
point of myristic acid je 54 °C, margaric (n-17:0) 61 °C,
while that of ai-margaric (ai-17:0) is a mere 37 °C. Hence a
change of branching from straight-chain to ai (e.g. C17 fatty
acid) the melting point drops by 24 °C, whereas chain
shortening of straight-chain from C17 to C14 (i.e. myristic
acid) only by 17 °C. Melting point of (Z)-15-methyl-hex-
adec-12-enoic acid (i-17:1) is 21–22 °C. We therefore
assume that fluidity change is affected much more by
branching than the shortening of FA.
0
to 18.1% (precursor Val). Precursor directed biosynthesis
based on i-Bu showed comparable results, see Table 2. The
overall proportion of even-i-FA increased to one quarter of
total FA as compared to control (cultivation on succinate as
the only carbon and energy source).
Apart from the increase in even-i-FA, an increased
content was also noted with odd-straight-chain FA, i.e.
1
5:0 and 17:0. This is probably due to the fact that one of
the possible products of Val (and also isobutyrate) catab-
olism is propionate, which serves as a starter unit of
odd-straight-chain FA. Analogous effect, i.e. increased
proportion of odd-straight-chain FA, was found in culti-
vations of other bacteria [10, 33].
Precursor directed biosynthesis of FA with the use of
Leu or i-Va as starter units leads to an increase in odd-i-FA
(
i-15:0 to 14.8% with Leu and 14.2% with i-Va, i-17:0 to as
much as 19.5% with Leu and 20.2% with i-Va). The pro-
portion of these FA rose to 35% of total FA. These results
are in agreement with the effect of both starter units
described in the literature [12, 34].
Ile
80
1
30
45
15
53
2
0
5
24
5
6
3
9
11
60
66
68
0
Leu
8
0
45
3
0
1
20
56
5
11
1
5
5
3
60
24
66
68
39
0
Val
8
0
45
1
30
60
5
20
5
6
1
1
15
66
2
4
5
3
39
68
7
0
0
5
0
60
70
80
90
100
time (min)
Fig. 12 RP-HPLC/APCI-MS chromatogram of the TAG from R. erythropolis cultivations at 30 °C with different carbon sources (Val, Leu, and
Ile) as determined by RP-HPLC/MS-APCI
1
23

7
54
Lipids (2010) 45:743–756
Cultivations in the presence of Leu and Ile and the
Table 4 Relative quantities (%) of TAG identified in R. erythropolis
cultivations at 30 °C with different carbon sources [succinate, Val,
Leu, Ile, isobutyric (i-Bu), isovaleric (i-Va) and 2-methyl-butyric
corresponding short FA (i-Va and 2-MeBu) did not show
any catabolism to acetate and propionate. Ile is known to
be catabolized to acetate and propionate, Leu to acetoac-
etate and acetate [35]. In the former case we can assume
that the starter units, i.e. acetate and propionate, have the
same influence on the biosynthesis of even and odd FA and
their contribution is thereby in fact eliminated (1 mol of
Leu or i-Va gives rise to 1 mol of acetate and propionate
and contributes thereby by the same molar amount to the
biosynthesis of even and odd FA).
(
2-MeBu) acid] as determined by RP-HPLC/MS-APCI
TAG
Succinate Val i-Bu Leu i-Va Ile
2-MeBu
Po/Po/O
brX/Po/Po
Po/Po/Mo
X/Po/Po
M/Po/O
brM/brM/O
Po/Po/P
M/Po/P
M/M/O
brM/brM/P
M/M/P
2.5
a
2.4 2.7
2.2 2.1
2.5 2.2
1.8 2.8
2.3 2.2
1.5 2.4
2.2 2.3
–
–
–
2.8 3.0
1.6 1.7
1.8 1.8
1.6 1.7
0.0
1.7
2.0
–
2.2 2.4
2.8 3.1
1.8 1.9
1.9 1.2
2.7 3.0
2.1 2.2
1.2 1.2
2.2 1.5
1.4 1.5
1.9 2.0
2.1 2.3
2.1 2.3
1.5 1.5
2.4 2.6
1.7 1.9
4.1 2.9
3.5 2.1
1.7 1.8
0.8 0.4
2.6 2.9
2.0 2.1
1.3 1.4
0.5 0.4
1.1 1.2
–
–
–
–
In the case of Ile (2-MeBu) only acetate and no
propionate is formed. Because the proportion of even FA
is major (more than 75% of total FA), an increase in the
content of the precursor, i.e. acetate, has no further effect
on the ratio of even and odd FA arising in the
biosynthesis.
2.1
1.6
1.5
–
2.6 3.4
2.0 2.9
0.9 1.6
2.1 2.1
1.5 1.5
1.0 1.0
–
–
–
–
1.1
0.8
0.7
3.8
3.3
3.4
1.2
–
1.4 2.1
1.6 2.3
2.1 2.9
1.9 2.7
1.3 1.9
2.4 3.2
1.5 0.3
0.8 0.9
1.8 1.8
1.6 1.7
2.4 2.4
1.8 1.8
2.2 2.3
1.1 1.4
Po/Mo/O
Po/P/Mo
Po/O/O
M/O/O
The results of the cultivation fully supported our
assumption that Rhodococcus is eminently suitable for
precursor directed biosynthesis of branched FA.
Chromatogram of TAG obtained after cultivation of
Rhodococcus with three amino acids (Val, Leu and Ile) is
shown in Fig. 12. Three pairs of mutually metabolically
coupled precursors were used - Val and i-Bu, Leu and i-Va,
Ile and 2-MeBu. The given amino acid gives rise to cor-
responding branched FA, which is then incorporated into
the molecule of appropriate FA.
Po/P/O
Po/Po/S
Po/brP/brP
M/brP/brP
M/P/O
–
–
–
–
–
–
–
–
–
2.9
–
1.8 2.1
1.6 1.6
brM/brM/S
Po/P/P
–
–
–
–
3.0
2.5
1.4
0.1
0.7
–
2.9 3.7
2.2 3.0
1.1 1.5
0.3 0.0
0.9 0.0
2.0 2.4
1.4 1.9
2.5 2.9
1.8 2.4
1.8 2.4
2.3 2.9
1.7 2.3
2.2 2.8
1.3 0.4
2.1 2.1
1.5 1.5
1.3 1.4
0.1 0.2
0.7 0.8
3.1 3.2
1.7 1.9
3.0 3.1
2.1 2.0
1.8 1.8
2.0 1.8
2.6 2.5
2.4 2.4
1.5 1.5
Table 4 gives mean (three independent sequential
measurements) relative peak areas of 69 TAG from Rho-
dococcus determined in control cultivation on succinate
and in individual cultivations using precursor directed
biosynthesis with six starter units. It should be stressed that
these values were calculated without using response fac-
tors, similarly as in our preceding paper in which we also
analyzed unusual plant TAG [36]. None of the standards is
commercially available and the 11 TAG that we synthe-
sized represent a small fraction (\1/6) of the total possible
TAG. Since the other standards are not available, the val-
ues are only semi-quantitative. This however does not
prevent comparison of the outcomes of individual culti-
vations because these comparisons involve structurally
similar TAG. An extrapolation of the response factors for
TAG differing only in the branched chain (e.g. brMa/S/O
and Ma/S/O) is unfortunately not feasible since we do not
know the differences between straight- and branched
chains. Despite this we attempted to quantify the results for
Ma/Ma/Ma, iMa/iMa/iMa, and aiMa/aiMa/aiMa, see
M/P/P
Mo/Mo/O
M/M/S
M/Po/S
brX/O/O
MoOO
–
–
3.2
–
1.5 1.6
brX/P/O
X/O/O
–
–
3.1
2.9
2.8
5.1
4.7
2.5
–
2.2 2.3
1.8 1.9
2.4 2.6
1.8 1.9
2.0 2.2
1.4 1.5
3.5 2.5
2.3 2.5
1.6 1.8
0.8 0.8
P/Mo/O
X/P/O
O/O/O
P/O/O
Po/S/O
brP/brP/O
P/P/O
–
–
–
–
4.3
2.1
1.9
–
2.6 3.3
1.8 0.8
0.7 0.1
2.4 1.8
1.4 0.0
2.3 2.2
1.4 1.4
0.9 0.9
3.3 3.2
2.2 2.4
Po/P/S
M/O/S
brMa/O/O
brX/O/S
brP/brP/P
Ma/O/O
X/O/S
–
–
–
–
–
‘
‘Experimental’’. If a value of 1.00 is arbitrarily allotted to
3.6 2.8
1.8 1.8
1.4 1.5
2.5 2.8
–
–
–
–
straight-chain TAG (Ma/Ma/Ma), the correction factor for
iso TAG is 0.97 and for ai-TAG 0.99. In the case of a real
sample, and also of several synthetic standards, we have
failed to separate TAG differing only in branching (i.e. iso
and anteiso).
3.4
0.0
3.9
–
1.2 1.1
1.2 0.8
3.1 3.8
2.9 2.4
1.8 1.8
1.2 1.1
2.1 2.1
3.2 3.0
P/P/P
P/brMa/O
–
–
1
23

Lipids (2010) 45:743–756
755
Table 4 continued
molecular species of TAG potentially produced by Rho-
dococcus and differing in branched chain FA is feasible.
The outcome of such analysis depends understandably on
the detection limit, which was in our case 0.1% total TAG.
We cannot exclude the possibility that under conditions
different from those used in this study, e.g. in stress-
inducing culture conditions or in biofilm, the proportions of
individual TAG will change. The sum of branched TAG
after precursor directed biosynthesis ranged from 21% for
the compound with the lowest incorporation, i.e. i-Bu, to
33% for Ile. These data strongly imply that Rhodococcus is
a good producer of branched chain FA and corresponding
TAG and can be successfully used as a biotechnologically
important microorganism producing these substances that
have considerable pharmacological potential.
TAG
Succinate Val i-Bu Leu i-Va Ile
2-MeBu
M/P/S
1.6
0.0
3.0
–
1.0 1.1
1.1 1.2
1.9 2.1
1.2 0.4
1.2 0.2
1.7 2.4
3.3 2.9
2.2 2.9
0.7 0.6
1.1 0.8
1.5 0.5
0.7 0.7
P/Mo/S
P/Ma/O
P/P/brMa
P/P/Ma
Po/S/S
0.9 0.9
1.7 1.6
3.0 2.9
1.5 1.5
0.6 0.7
1.7 1.6
1.5 1.5
–
–
–
2.2 2.4
0.7 0.8
1.1 1.1
1.3 1.4
2.7 1.8
1.5 1.7
1.1
3.7
3.4
–
O/O/S
P/O/S
brP/brP/S
P/P/S
–
0.0
2.0 0.4
3.5 2.0
0.1 0.1
1.2 2.0
1.8 0.5
0.6 0.4
2.2 1.2
0.3 1.2
0.1 0.1
1.1 0.6
0.5 0.3
0.9 0.2
1.2 0.2
0.1 0.3
–
0.0
3.0
–
1.4 1.3
3.9 3.7
0.0 0.0
0.9 0.9
2.4 2.3
0.9 0.9
2.3 2.1
0.3 0.3
0.2 0.2
0.8 0.8
0.8 0.8
0.7 0.6
1.5 1.4
0.1 0.0
P/brMa/brMa
M/S/S
–
–
0.6
0.1
–
0.1 0.0
2.0 2.0
In conclusion, RP-HPLC/MS-APCI made it possible to
identify and quantify TAG differing in a single branched-
chain FA. Branched- and straight-chain-TAG were sepa-
rated and identified while TAG differing only in iso- or
anteiso-FA could not be separated since their APCI mass
spectra were completely identical. Branched TAG having
two branched FA in the molecule were successfully
separated from straight-chain counterparts by gradient
elution. TAG having a single branched FA were sepa-
rated only if they differed in the site of branching.
Positional isomers such as P/P/Ma and P/Ma/P or P/Ma/
Ma and Ma/P/Ma were partially separated only with
isocratic elution.
P/Ma/Ma
brMa/O/S
Ma/O/S
P/brMa/S
Ma/Ma/Ma
Mo/S/S
P/Ma/S
O/S/S
–
–
2.0
–
1.1 1.0
–
–
–
–
1.6
0.4
0.3
2.4
2.0
–
0.1 0.1
1.3 1.3
0.4 0.4
0.6 0.7
P/S/S
brMa/S/S
Ma/S/S
a
–
–
0.2
0.3 0.3
Minority TAGs up to 0.1% of total TAGs were omitted
The two-sample paired t test revealed that a significant
incorporation of precursors into the molecules of TAG
appeared only in the pair Val and i-Bu (P value is 0.012).
This value of the t test shows that at 5% significance level
we reject the hypothesis of zero differences. The values for
the other two pairs, i.e. Leu and i-Va and Ile and 2-MeBu,
are different (P value is 0.154 and 0.453, respectively).
This feature can be demonstrated in the ensuing pair of
TAG, i.e. M/M/O and iM/iM/O. Precursors such as Val or
i-Bu cannot give rise to ai-FA. Based on the t test we can
thus infer that more Val was incorporated in branched TAG
than i-Bu, because the ratio of Val:i-Bu incorporation in
the former species was 1.2:1.2 while in branched TAG it
was 1.9:1.2. Further confirmation of this fact is seen in
Table 4. Another feature distinguishing the incorporation
of Val (i-Bu) from that of the other two pairs (Leu/i-Va and
Ile/2-MeBu) into the molecular species of TAG is the
presence of only those molecular species that have two
branched FA in the molecule, whereas with Leu and Ile
Acknowledgments This work was supported by the Institutional
Research Concepts AV0Z50200510, projects MSM6046137305 and
AROMAGEN—2B08062.
References
1
. Waltermann M, Steinbuchel A (2005) Neutral lipid bodies in
prokaryotes: recent insights into structure, formation, and rela-
tionship to eukaryotic lipid depots. J Bacteriol 187:3607–3619
. Alvarez HM, Steinbuchel A (2002) Triacylglycerols in prokary-
otic microorganisms. Appl Microbiol Biotechnol 60:367–376
. Alvarez HM, Mayer F, Fabritius D, Steinbuchel A (1996) For-
mation of intracytoplasmic lipid inclusions by Rhodococcus
opacus strain PD630. Arch Microbiol 165:377–386
2
3
4
5
. Waltermann M, Luftmann H, Baumeister D, Kalscheuer R,
Steinbuchel A (2000) Rhodococcus opacus strain PD630 as a new
source of high-value single-cell oil? Isolation and characteriza-
tion of triacylglycerols and other storage lipids. Microbiology
146:1143–1149
. Hernandez MA, Mohn WW, Martinez E, Rost E, Alvarez AF,
Alvarez HM (2008) Biosynthesis of storage compounds by
Rhodococcus jostii RHA1 and global identification of genes
involved in their metabolism. BMC Genomics 9:600
(
and the corresponding short-branched acids) the only
identified molecular species of TAG were those having
only one branched FA in the molecule. Based on the
chromatographic behavior of the standards we synthesized
we can confirm that the separation and identification of the
6. Alvarez HM, Luftmann H, Silva RA, Cesari AC, Viale A,
Waltermann M, Steinb u¨ chel A (2002) Identification of phenyl-
decanoic acid as a constituent of triacylglycerols and wax ester
produced by Rhodococcus opacus PD630. Microbiology
148:1407–1412
1
23

7
56
Lipids (2010) 45:743–756
7
8
9
. Hamilton JTG, McRoberts WC, Larkin MJ, Harper DB (1995)
Long-chain haloalkanes are incorporated into fatty acids by
22. Cejkova A, Masak J, Jirku V, Vesely M, Patek M, Nesvera J
(2005) Potential of Rhodococcus erythropolis as a bioremediation
organism. World J Microbiol Biotechnol 21:317–321
Rhodococcus rhodochrous NCIMB 13064. Microbiology
1
41:2611–2617
23. Rezanka T, Mares P (1987) Unusual and very long-chain fatty-
acids produced by Basidiomycetes. J Chromatogr 409:390–395
24. Rezanka T (1990) Identification of very long polyenoic acids as
. Alvarez HM, Kalscheuer R, Steinbuchel A (1997) Accumulation
of storage lipids in species of Rhodococcus and Nocardia and
effect of inhibitors and polyethylene glycol. Fett/Lipid
?
picolinyl esters by Ag ion-exchange high-performance liquid
chromatography, reversed-phase high-performance liquid chro-
matography. J Chromatogr 513:344–348
9
9:239–246
. Bode HB, Dickschat JS, Kroppenstedt RM, Schulz S, Muller R
2005) Biosynthesis of iso-fatty acids in Myxobacteria: iso-even
(
25. Ziegler FE, Berger GD (1979) A mild method for the esterifi-
cation of fatty acids. Synth Commun 9:539–543
1
26. Lie Ken Jie MSF, Lam CC (1995) H-Nuclear magnetic reso-
fatty acids are derived by alpha-oxidation from iso-odd fatty
acids. J Am Chem Soc 127:532–533
1
1
0. Jovetic S, Feroggio M, Marinelli F, Lancini G (2008) Factors
influencing cell fatty acid composition, A40926 antibiotic com-
plex production in Nonomuraea sp. ATCC 39727. J Ind Micro-
biol Biotechnol 35:1131–1138
1. Dherbecourt J, Maillard MB, Catheline D, Thierry A (2008)
Production of branched-chain aroma compounds by Propioni-
bacterium freudenreichii: links with the biosynthesis of mem-
brane fatty acids. J Appl Microbiol 105:977–985
nance spectroscopic studies of saturated, acetylenic and ethylenic
triacylglycerols. Chem Phys Lipids 77:155–171
27. Momchilova S, Itabashi Y, Nikolova-Damyanova B, Kuksis A
(2006) Regioselective separation of isomeric triacylglycerols by
reversed-phase high-performance liquid chromatography: sta-
tionary phase and mobile phase effects. J Sep Sci 29:2578–2583
28. Dinh-Nguyen N (1968) Contribution to the study of mass spec-
trometry m. 3: use of methyl esters of normal long-chain fatty
acids labelled with deuterium or C-13. Ark Kemi 28:289–362
29. Mottram HR (2005) Regiospecific analysis of triacylglycerols
using high performance liquid chromatography/atmospheric pres-
sure chemical ionization mass spectrometry. In: Byrdwell WC (ed)
Modern methods for lipid analysis by liquid chromatography/mass
spectrometry and related techniques, AOCS Press, Champaign
30. Byrdwell WC (2005) Qualitative and quantitative analysis of
triacylglycerols by atmospheric pressure ionization (APCI & ESI)
techniques. In: Byrdwell WC (ed) Modern methods for lipid
analysis by liquid chromatography/mass spectrometry and related
techniques. AOCS Press, Champaign
31. Fauconnot L, Hau J, Aeschlimann JM, Fay LB, Dionisi F (2004)
Quantitative analysis of triacylglycerol regioisomers in fats and
oils using reversed-phase high-performance liquid chromatogra-
phy and atmospheric pressure chemical ionization mass spec-
trometry. Rapid Commun Mass Spectrom 18:218–224
32. Leskinen H, Suomela JP, Kallio H (2007) Quantification of tri-
acylglycerol regioisomers in oils and fat using different mass
spectrometric and liquid chromatographic methods. Rapid Com-
mun Mass Spectrom 21:2361–2373
33. Dickschat JS, Bode HB, Kroppenstedt RM, Muller R, Schulz S
(2005) Biosynthesis of iso-fatty acids in myxobacteria. Org
Biomol Chem 2824–2831
34. Zhu K, Bayles DO, Xiong A, Jayaswal RK, Wilkinson BJ (2005)
Precursor and temperature modulation of fatty acid composition
and growth of Listeria monocytogenes cold-sensitive mutants
with transposon-interrupted branched-chain a-keto acid dehy-
drogenase. Microbiology 151:615–623
35. Massey LK, Sokatch JR, Conrad RS (1976) Branched-chain
amino-acid catabolism in bacteria. Bacteriol Rev 40:42–54
36. Lisa M, Holcapek M, Rezanka T, Kabatova N (2007) HPLC/
APCI-MS and GC/FID characterization of 5-olefinic fatty acids
1
1
1
2. Beck HC (2005) Branched-chain fatty acid biosynthesis in a
branched-chain amino acid aminotransferase mutant of Staphy-
lococcus carnosus. FEMS Microbiol Lett 243:37–44
3. Beck HC, Hansen AM, Lauritsen FR (2004) Catabolism of leu-
cine to branched-chain fatty acids in Staphylococcus xylosus.
J Appl Microbiol 96:1185–1193
4. Vlaeminck B, Fievez V, Cabrita ARJ, Fonseca AJM, Dewhurst
RJ (2006) Factors affecting odd- and branched-chain fatty acids
in milk: a review. Anim Feed Sci Technol 131:389–417
5. Parodi PW (2005) Dairy product consumption and the risk of
breast cancer. J Am Coll Nutr 24:556S–568S
6. Wongtangtintharn S, Oku H, Iwasaki H, Toda T (2004) Effect of
branched-chain fatty acids on fatty acid biosynthesis of human
breast cancer cells. J Nutr Sci Vitaminol 50:137–143
1
1
1
1
1
2
2
7. Rezanka T, Schreiberova O, Krulikovska T, Masak J, Sigler K
(2010) RP-HPLC/MS-APCI analysis of odd-chain TAG from
Rhodococcus erythropolis including some regioisomers. Chem
Phys Lipids 163:373–380
8. Metz PA, Omstead DR, Kaplan L, Liesch JM, Stearns RA,
Vandenheuvel WJA (1988) Characterization of a lipid-rich
fraction synthesized by Streptomyces avermitilis. J Chromatogr A
4
41:31–44
9. Myher JJ, Kuksis A, Marai L (1993) Identification of the less
common isologous short-chain triacylglycerols in the most vol-
atile 2.5% molecular distillate of butter oil. J Am Oil Chem Soc
7
0:1183–1191
0. Mottram HR, Evershed RP (2001) Elucidation of the composition
of bovine milk fat triacylglycerols using high-performance liquid
chromatography-atmospheric pressure chemical ionisation mass
spectrometry. J Chromatogr A 926:239–253
1. Marai L, Kuksis A, Myher JJ (1994) Reversed-phase liquid
chromatography-mass spectrometry of the uncommon triacyl-
glycerol structures generated by randomization of butteroil.
J Chromatogr A 672:87–99
in triacylglycerols from conifer seed oils.
1146:67–77
J Chromatogr
1
23