Chemo-enzymatic synthesis of novel β-hydroxy-β-methylbutryric acid (HMB)-medium chain triacylglycerol (MCT) complexes

Article abstract of DOI:10.1007/s11746-013-2220-5

β-Hydroxy-β-methylbutyrate (HMB) is reported to have ergogenic benefits such as reducing muscle wasting, promoting exercise performance and skeletal muscle hypertrophy. When used as a dietary feed, it has been found to improve colostral milk fat and the growth of piglets. Medium chain triacylglycerols (MCT) are a good energy source as they can be subjected to β-oxidation in the liver without being stored as fats. When used as animal feed, HMB-MCT complexes are reported to stimulate growth and produce leaner meat product. A high yield two-steps chemo-enzymatic pathway for synthesis of HMB-MCT complexes has been developed. Ethyl HMB ester was first synthesized using a chemical catalyst. Following that, it was transesterified with MCT by using a biocatalyst. Approximately 85 wt% of HMB-MCT complexes can be achieved using this two-step synthetic pathway. The reaction mechanism and some of the reaction parameters were also briefly elucidated.

Full text of DOI:10.1007/s11746-013-2220-5

J Am Oil Chem Soc (2013) 90:919–922
DOI 10.1007/s11746-013-2220-5
LETTER TO THE EDITOR
Chemo-enzymatic Synthesis of Novel b-Hydroxy-b-methylbutryric
Acid (HMB)–Medium Chain Triacylglycerol (MCT) Complexes
•
Ling-Zhi Cheong Katarzyna Widzisz
•
•
Yingyao Wang Henrik Helligsø Jensen
•
•
Peter Kappel Theil Zheng Guo Xuebing Xu
•
Received: 20 January 2012 / Revised: 19 June 2012 / Accepted: 18 February 2013 / Published online: 15 March 2013
Ó AOCS 2013
Abstract b-Hydroxy-b-methylbutyrate (HMB) is repor-
ted to have ergogenic benefits such as reducing muscle
wasting, promoting exercise performance and skeletal
muscle hypertrophy. When used as a dietary feed, it has
been found to improve colostral milk fat and the growth of
piglets. Medium chain triacylglycerols (MCT) are a good
energy source as they can be subjected to b-oxidation in the
liver without being stored as fats. When used as animal
feed, HMB–MCT complexes are reported to stimulate
growth and produce leaner meat product. A high yield two-
steps chemo-enzymatic pathway for synthesis of HMB–
MCT complexes has been developed. Ethyl HMB ester was
first synthesized using a chemical catalyst. Following that,
it was transesterified with MCT by using a biocatalyst.
Approximately 85 wt% of HMB–MCT complexes can be
achieved using this two-step synthetic pathway. The reac-
tion mechanism and some of the reaction parameters were
also briefly elucidated.
Dear Editor,
b-Hydroxy-b-methylbutyrate (HMB) is a metabolite of the
essential branched-chain amino acid leucine, which has
been found to reduce muscle wasting in patients associated
with trauma and cancer cachexia by reducing muscle
atrophy and increasing muscle hypertrophy [1, 2]. In
addition, HMB has also been reported to have ergogenic
benefits including anticatabolic, anabolic and lipolytic
effects. As a result, HMB in the form of the calcium salt
has been used extensively by bodybuilders and athletes as
an ergogenic acid to promote exercise performance and
skeletal muscle hypertrophy [3]. Although not studied
extensively yet, HMB when incorporated into dietary feed
was found to result in improved colostral milk fat and
growth of piglets [4]. Medium chain triacylglycerols
(MCT) such as tricaprylin and tricaprin are reported to be
readily hydrolyzed by lingual and gastric lipases. The
medium chain fatty acids formed are then subjected pre-
dominantly to b-oxidation in the liver without being stored
as fats. Thus, MCT is a good energy source especially for
patients with pancreatic insufficiency and fat malabsorp-
tion [5]. In addition, MCT improves colostrum yield of
sows and thus ensure a higher transfer of nutrients to the
piglets immediately after birth [6]. Taking these beneficial
effects into consideration, HMB if incorporated into MCT
is postulated to have combined beneficial effects of an
increased muscle growth rate and decreased accumulation
of fat. This may present an interesting opportunity to the
animal feed industry as such structured lipids are expected
to improve the animal growth rate and meet the current
demand of health-conscious consumers for leaner meat
products. In addition, a structured lipid is regarded as a fat
source and can be added to animal feed and does not
require approval for inclusion in animal feeds. To date,
L.-Z. Cheong (&) Á K. Widzisz Á Y. Wang Á Z. Guo Á X. Xu
Department of Engineering, Aarhus University,
8000 Aarhus, Denmark
e-mail: cheong@mb.au.dk
Y. Wang
Academy of State Administration of Grain, Beijing, China
H. H. Jensen
Department of Chemistry, Aarhus University,
8000 Aarhus, Denmark
P. K. Theil
Department of Animal Science, Aarhus University,
8830 Tjele, Denmark
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J Am Oil Chem Soc (2013) 90:919–922
there are no, or only limited, studies that examine either the
benefits effects of or the synthetic pathway of HMB–MCT
complexes. The present study was aimed at developing
an efficient pathway for a high yield of HMB–MCT
complexes.
the wt% of the total weight of the sample. Standard curves
were constructed using pure reaction substrates (ethyl
HMB esters and tricaprylin) and purified reaction products.
The one-step enzymatic acidolysis of HMB and trica-
prylin was carried out for 5 days. Nevertheless, no enzy-
matic activity or product formation was observed in the
one-step enzymatic acidolysis of HMB and tricaprylin. This
is mainly due to the strong acidity of HMB which disrupts
the enzyme structure and causes it to lose its catalytic
activity. Numerous studies have reported that the presence
of strong acid such as acetic acid will affect the aqueous
microenvironment of the biocatalyst resulted in inactivation
of lipase. Novozym 435; for example, was found to be most
catalytically active at neutral pH of 7 to 8. It was shown that
activity of Novozym 435 dropped tremendously in both
acidic and alkaline microenvironments [7]. The reaction
mixture containing both HMB and tricaprylin was rather
acidic with a pH of approximately 3 to 4. Thus, this may
have resulted in inactivation of Novozym 435.
A one-step enzymatic acidolysis of HMB and tricaprylin
was first attempted to produce HMB–MCT complexes.
HMB purchased from Th. Geyer Danmark (Roskilde,
Denmark) and tricaprylin obtained from Cognis Care
Chemicals, Dusseldorf, Germany) were weighed accurately
at different substrate molar ratios [3:1 (2.67:3.53 mg), 2:1
(1.78:3.53 mg), 1:1 (0.89:3.53 mg), 1:2 (0.89:7.06 mg) and
1:3 (0.89:10.59 mg)] into jacketed glass reactors and stir-
red at 250 rpm. The jacketed glass reactors were then
heated to a constant 65 °C using a circulating water bath.
The acidolysis reactions were initiated by addition of
Novozym 435 (10 wt% of total substrate weight).
Novozym 435 (lipase from Candida antarctica B) was
kindly donated by Novozymes A/S (Bagsværd, Denmark).
The reactions were carried out in duplicate. Reactions were
monitored by withdrawing the reaction mixtures periodi-
cally for detection of changes in acylglycerol composition
throughout the duration of the reaction. Analysis was
carried out using a high performance liquid chromato-
graph (HPLC) (Thermo Fisher Scientific Inc., Roskilde,
Denmark). Separation of the different components was
performed using a reverse phase Supelcosil LC-18, 5 lm
column (250 mm x 4.6 mm) (Supelcosil Inc., Bellefonte,
PA). A binary solvent system of acetonitrile (solvent A)
and isopropanol: hexane (2:1) (solvent B) under gradient
elution was used. Beginning with 70 % solvent A and
30 % solvent B, solvent A was reduced to 40 % and sol-
vent B was increased to 60 % at 40 min. The composition
of 40 % solvent A and 60 % solvent B was maintained for
10 min before reverting back to 70 % solvent A and 30 %
solvent B for another 6 min. The flow rate of the solvent
was at a constant 1.0 ml/min. A Sedex (S.E.D.E.R.E.,
Alfortville, France) model 75 ELSD was used for detec-
tion; the pressure of the nebulizer gas (air) was maintained
at 3.2 bar and the drift tube temperature was set at 40 °C.
The identity of each peak was identified by determining its
molecular mass using a Dionex Ultimate 3000 HPLC
system coupled through an electrospray ionization (ESI)
inlet to a qTOF mass spectrometer (microTOFq) (Bruker
Daltonic GmbH, Bremen, Germany). Data were acquired
and processed using Bruker Compass software including
Bruker Daltonics Hystar, MicroTOF control and Data-
analysis. Samples were analyzed using both negative and
positive ionization mode to ensure detection of all relevant
compounds. The ESI-qTOF was operated under the fol-
lowing conditions: nebulizer pressure at 3.4 bar, dry gas
flow 10 L/min, source voltage 4.0 kV and transfer time
120 ms. The acylglycerol composition was expressed as
Following the ineffective single-step synthetic pathway,
we proposed a two-step chemo-enzymatic pathway to
synthesize HMB–MCT complexes. In the first step, the
acidity of HMB was reduced by converting HMB into ethyl
HMB ester through esterification (Fig. 1a). In the second
step, ethyl HMB ester was transesterified with tricaprylin to
produce HMB–MCT complexes (Fig. 1b).
Esterification of HMB was conducted by using chemical
catalysts. Two chemical catalysts were studied namely
sulfuric acid and p-toluenesulfonic acid anhydrous. Sulfu-
ric acid was obtained from Sigma Aldrich (Brøndby,
Denmark); meanwhile, p-toluenesulfonic acid anhydrous
was purchased from Eurolabs Ltd. (Cheshire, United
Kingdom). Firstly, HMB (8.85 g), absolute ethanol (6.9 g)
[molar ratio of HMB:ethanol, 1:2] and chemical catalyst
(5 wt% of HMB) were weighed accurately into a 100-ml
round-bottom flask. In the case where p-toluenesulfonic
acid anhydrous was used, 45 ml of cyclohexane and 70 g
of molecular sieves were also added to the reaction flask.
The reaction was initiated by heating the mixture con-
stantly under reflux at 85 °C by using an oil bath. After 5 h
of reaction, the reaction was stopped by cooling the reac-
tion mixture to room temperature and adding anhydrous
sodium sulfate. The reaction mixture was then distilled at
150 mbar and 50 °C to remove the cyclohexane and excess
ethanol. Finally, the resulting distillate was washed several
times using a mixture of dichloromethane and saturated
aqueous sodium bicarbonate (1:1/v:v). Following the
washing steps, the mixture was centrifuged to obtain the
organic and aqueous phases. The organic phase was pooled
and distilled to obtain ethyl HMB ester. The purity and
molecular structure of the ethyl HMB ester was identified
1
by H NMR performed on a Varian Mercury 400 MHz
NMR spectrometer (Palo Alto, CA, USA).
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J Am Oil Chem Soc (2013) 90:919–922
921
(A)
O
O
Catalyst
HO
CH3
+
H2O
HO
CH3
+
OH
Ethanol
O
OH
HMB
Water
Ethyl HMB Ester
(B)
O
HO
CH3
+
Tricaprylin
O
O
O
O
Ethyl HMB Ester
O
O
O
Catalyst
Product 2
(2-caprylyl-1,3-di-hydroxy-methyl-butyrin)
+
O
O
O
O
O
O
O
O
O
H3C
HO
O
CH3
OH
Product 1
(1-hydroxy-methylbutyl-2,3-dicaprylin
or 3-hydroxy-methylbutyl-1,2-dicaprylin)
CH3
O
O
OH
Fig. 1 The two-step chemoenzymatic pathway for high yield synthesis of HMB–MCT complexes. a Step 1: esterification of HMB to form ethyl
HMB esters. b Step 2: transesterification of ethyl HMB ester with tricaprylin to form HMB–MCT complexes
In the esterification reaction when sulfuric acid was used
as the catalyst, a very low yield of ethyl HMB ester (10 %)
was obtained with the formation of many unwanted side
products. Sulfuric acid is a strong acid catalyst; thus has the
capability of reacting with both the carboxylic and hydro-
xyl groups of the HMB. In addition, as molecular sieves
were not used in the reaction catalyzed by sulfuric acid,
there is a possibility that water molecules formed during
the reaction further hydrolyzed the products forming
unwanted side products. In an attempt to increase the yield
of ethyl HMB esters, sulfuric acid was replaced with
p-toluensulfonic acid anhydrous as catalyst. In addition,
cyclohexane and molecular sieves were also added to the
reaction mixture. This reaction proved itself to be more
efficient with 80 % of purified ethyl HMB esters obtained
following the purification step. The cyclohexane, ethanol,
and water mixture formed an azeotrope which resulted in
a lower evaporation temperature. As a result, the water
molecules formed can be effectively absorbed by the
molecular sieves without causing unwanted hydrolysis of
the ethyl HMB ester. The structure of the purified ethyl
2.41 (s, 2H, CH2C(O)), 1.21 (s, 6H, CH3), 1.21 (t, 3H,
OCH2CH3).
The ethyl HMB ester obtained was then transesterified
with tricaprylin using lipase Novozym 435. Ethyl
HMB esters and tricaprylin at different molar ratio [3:1
(3.29:3.53 mg), 2:1 (2.19:3.53 mg), 1:1 (1.10:3.53 mg),
1:2 (1.10:7.06 mg) and 1:3 (1.10:10.59 mg)] were weighed
accurately into jacketed glass reactors and stirred at
250 rpm. The jacketed glass reactors were then heated
constantly to 65 °C using a circulating water bath. The
acidolysis reactions were initiated by the addition of
Novozym 435 (10 wt% of substrate). Two different prod-
ucts were formed, namely product 1 with HMB being
incorporated at sn-1 or sn-3 position (1-hydroxy-meth-
ylbutyl-2,3-dicaprylin or 3-hydroxy-methylbutyl-1,2-dica-
prylin) (MW:429.29) and product 2 with HMB being
incorporated at sn-1 and sn-3 (2-caprylyl-1,3 di-hydroxy-
methylbutyrin) (MW:418.26) positions.
In order to develop an efficient synthesis method for a
high yield of HMB–MCT complexes, the effects of the
substrate molar ratio were examined. As expected, HMB–
MCT complexes increased with the amount of ethyl HMB
esters (Fig. 2a). After 72 h of reaction, approximately
1
HMB ester was confirmed by using H NMR. 1H NMR
(CDCl3, 400 MHz) dH 4.12 (q, 2H, J 7.2 Hz, OCH2CH3),
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J Am Oil Chem Soc (2013) 90:919–922
80
70
60
50
40
30
20
10
0
product 2 started to increase. This is mainly due to the
increasing amount of product 1 in the reaction pool which
prompted it to act as a substrate for transesterification with
tricaprylin forming product 2.
(A)
Product 1
Product 2
In summary, a high-yield two-step chemo-enzymatic
pathway for the synthesis of HMB–MCT complexes has
been developed. Ethyl HMB ester was first synthesized
using a chemical catalyst. Subsequently, it was transeste-
rified with MCT by using a biocatalyst. Approximately
85 wt% of HMB–MCT complexes (66 wt% of product 1
and 19 wt% of product 2) can be achieved within 72 h
using this two-step synthetic pathway.
3:1
2:1
1:1
1:2
1:3
-10
Substrate Molar Ratio (Ethyl HMB Ester:Tricaprylin)
100
80
60
40
20
0
Unreacted substrates
Product 1
(B)
Product 2
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0
20
40
60
80
100
120
-20
Reaction time (h)
Fig. 2 Transesterification of ethyl HMB ester with tricaprylin.
a Effects of substrate molar ratio on transesterification b Time course
of the transesterification
85 wt% of HMB–MCT complexes was synthesized with
66 wt% of product 1 and 19 wt% of product 2 when the
molar ratio of ethyl HMB ester to tricaprylin was 3 to 1.
In order to further elucidate the reaction mechanism, the
aforementioned reaction was carried out for 120 h. As
shown in Fig. 2b, the reaction reached equilibrium at
approximately 60 h. The rate and extent for the syntheses
of product 1 was higher as compared to those of product 2.
Formation of product 1 reached a maximum at equilibrium
(60 h). After that, it started to decline and meanwhile
7. Romero MD, Calvo L, Alba C, Daneshfar A (2007) A kinetic
study of isoamyl acetate synthesis by immobilized lipase-catalyzed
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