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

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

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

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

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

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

4

CaCl ꢀ2H O 0.00026; (NH ) SO 0.71; FeSO ꢀ7H O 0.0006;

2

4 2

4

2

MgCl ꢀ6H O 0.34; Na MoO ꢀ2H O 0.002; pH was adjusted to

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

Ma/3.15

iMa/3.15

Ma/Ma/Ma

iMa/iMa/iMa

92

90

C99

[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

-Monopalmitin

,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

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 = a_M_a_/_M_a_/_M_a/a_T_A_G,

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

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

44:0

51:3

49:2

52:3

50:2

48:1

46:0

829

789

815

789

803

777

803

777

751

843

817

857

831

805

779

575

535

561

535

575

549

523

589

521

603

575

577

549

551

Po/Po

M/Po

547

521

495

547

523

495

561

575

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

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/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

3

4

5

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

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

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

54:3

52:2

50:1

53:2

51:1

48:0

53:2

51:1

48:0

51:1

48:0

51:1

49:0

52:1

54:2

52:1

50:0

51:0

50:0

51:0

50:0

53:1

51:0

53:1

51:0

54:1

52:0

53:0

805

779

805

779

857

779

805

819

871

845

819

845

885

859

833

873

847

807

873

847

807

847

807

805

847

821

861

887

861

835

849

835

849

835

875

849

875

849

889

863

877

P/O

577

551

549

551

589

551

577

603

577

603

589

577

603

605

577

579

605

603

605

551

603

605

551

577

579

591

577

565

607

605

579

607

579

605

565

579

607

565

605

579

607

523

495

551

523

575

495

521

563

589

537

563

521

537

603

577

551

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

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

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

577

603

577

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

–

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

579

–

S/O

Ma/O

brMa/P

Ma/Ma

S/S

P/S

–

Mo/S

Ma/S

S/S

591

593

607

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

3

60

24

66

68

39

0

Val

8

0

45

1

30

60

5

20

5

6

1

15

66

2

4

5

3

39

68

7

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

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

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

0.7 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

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

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

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

Article Doi

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

DOI: 10.1007/s11745-010-3447-7

Source and publish data:

Authors:

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

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

Products guided by the article

R&D Labs maybe for 122-32-7

Relevant to this article

Hot Product