LEVELS OF THIAMINE AND ITS ESTERS IN BEE COLLECTED POLLEN
USING LIQUID CHROMATOGRAPHY AND ROBOTICS
E.W. HERBERT, Jr. J.T. VANDERSLICE MEIS-HSIA HUANG * D.J. HIGGS U.S.
Department of Agriculture, Agricultural
ResearchService,
*
Beneficial
InsectsLaboratory,
and ** HNRC, NutrientComposition Laboratory, Beltsville,
MD 20705SUMMARY
The levels of thiamine
(B 1 )
and its mono- anddiphosphate
forms were determined in freshpollen
collected from
free-flying
colonies ofhoney
bees(Apr.-Oct. 1985)
located atBeltsville,
MD. The entireanalytical
process wasperformed by
alaboratory
robotic system.The seasonal thiamine levels
(w
moles/100g)
in bee-collectedpollen
variedgreatly depending
on thefloral source and time of year. All three forms of this vitamin are present in the
pollen samples
butthiamine is the
principle
form of the vitamin. The second most abundant form was thediphosphate
followed
by
themonophosphate
form.INTRODUCTION
Pollen, the high protein and vitamin diet of honey bees, is
arich
sourceof B-vitamins (H AYDAK and V IVINO , 1943 ; K OCK and S CHWARZ , 1956 ; and L
UNDEN
, 1954). V IVINO and P ALMER (1944) reported that of the B-vitamins, pantothenic acid, nicotinic acid, thiamine and riboflavin plus ascorbic acid
werepresent in the greatest
amounts.The levels of thiamine (B I ) in bee-collected pollen have been reported by
several investigators (V IVINO and P ALMER , 1944 ; H AGEDORN and BURGER,
1968). Although the precise function of thiamine in honey bees is not known, it may have a specific metabolic function in the oxidation of carbohydrates. It
forms part of
anenzyme system which is necessary for the decarboxylation of pyruvic acid,
anintermediate compound of carbohydrate metabolism. In
biological samples, vitamin B 1 activity is due not only
to thiamine but also
to
the mono, di and triphosphate derivatives of thiamine. Thiamine and its
mono-and diphosphate forms
arenormally the most prevalent forms. Pollen appa-
rently supplies the honey bees requirement for this vitamin since there is
noreal evidence that it
canbe synthesized by insects. Thiamine is relatively stable
in acid pH and pollen provides an acid environment (H ERBERT and S HIMANUKl , 1978). In stored pollen the vitamin has been shown
to exist for up
to 4 years
(H
AGEDORN and BURGER, 1968).
The present investigation was conducted
to determine the forms of thiamine and
to quantify their
amounts in pollen samples collected from free-
flying colonies of honey bees (Apr.-Oct. 1985) located at Beltsville, MD. The entire analytical process
was performed by
a laboratory robotic system prog- rammed
to perform the necessary extraction steps, the injection into the sample purification system, the collection of the effluent and the injection into
the final analytical system.
MATERIALS AND METHODS
Front-mounted
pollen traps
wereplaced
on hives offree-flying
colonies ofhoney
bees located atBeltsville, Maryland.
Freshpollen
was collectedweekly
fromApril
to October 1985. Allsamples
werefrozen at - 10 °C until the determinations were made.
The vitamins were extracted from bee
pollen
with aprocedure
similar to thatemployed
for vitaminB
6 (V ANDERSLICE
eta l ., 1980).
Ten ml of 5 %(w/v) sulfosalicylic
acid(SSA)
and 10 ml of hexane wereadded to a
sample
tubecontaining
1 g ofpollen
and a known amount of the internal standardamprolium (V
ANDERSLICE
andH UANG , 1986).
The mixture was mixed for oneminute, centrifuged
at 2400 g for 10minutes,
the waterlayer
removed and filteredthrough
a0.45 disposable filter,
and 0.5 ml of the filtrate wasinjected
into asample clean-up
system.The sole purpose of the
sample cleanup system
is to remove SSA from the filtrate. This is because SSA fluorescesstrongly
and interferes with thequantitative analysis.
Thecleanup
system is asimple high performance liquid chromatograph (HPLC)
systemconsisting
of pump,injection valve,
commercial anionexchange
column(packed
with 200-400 Bio-Rad AG2-X8 anionexchange resin)
and ultraviolet detector(Hitachi
Model 100-10Spectrophotometer).
The vitamersbegin
to elute after 1.5 min and 4.3 ml of effluent is collected. This effluent contains all of the vitamersplus
the internalstandard, amprolium.
A0.1 ml
aliquot
is theninjected
into the finalanalytical
HPLCsystem
which is described elsewhere(V
ANDERSLICE
and HUANG,1986).
This is a reversephase chromatographic
system which separates the various vitamers from one another and from the internal standard.Upon
elution from the column, the differentspecies
are oxidized to form thiochrome derivatives which are monitoredby
the fluorescence detector. The relative areas under the observedpeaks
areintegrated
and the concentration is automati-cally
determined from the known amount of internal standardorginally
added to thesample.
The
Laboratory
robotic system used in thisstudy
is shown inFig.
1. The robotic system consists ofan arm that can move in three dimensions and has two
interchangeable multipurpose
hands whichperforms
such acts ascapping, mixing, spinning, injecting
and a module whichprovides
communication between the robotic system and other instruments used in theanalytical procedure.
This robot can beprogrammed
toperform analyses continuously
over atwenty-four
hourperiod.
Theoperator
needonly
toprepare the
samples
and reagents and add internal standard to theweighed sample
in thesample
tubes.RESULTS
A typical chromatogram for
amixture of standards and for
an extractfrom bee pollen is shown in Fig. 2. The elution times
arethe
samefor both
standard mixture and pollen
extract.Also,
nopeaks which interfere with the determination of thiamine, thiamine monophosphate and thiamine diphosphate
are
present which indicates that the extraction and cleanup column
areyielding
«
clean
»samples for analysis.
The seasonal thiamine levels (!L moles/ 100 g) in bee collected pollen are
presented in Fig. 3. All three forms of this vitamin
are present in the pollen samples but thiamine is the principle form of the vitamin. The second
mostabundant form
wasthe diphosphate followed by the monophosphate form.
Pollen samples contained the greatest
amountsot the vitamin in early spring (12 Apr. collection), late May — early Jun (31 May and 7 Jun collection) and early fall (4 Oct. collection). The level apparently depends on the time of year and floral
source. For the
most part, the variation in the level of total vitamin is due
to the variation in thiamine
content as the levels of the monophosphate
and diphosphate forms remain relatively
constantthroughout the
season.To be
certain that the variation
was notdue
tomoisture variation the pollen samples
were vacuum
dried at 80 °C overnight and the total vitamin values
on adry weight basis are also shown in Fig. 3.
As noted in Fig. 3, thiamine values
are notpresented between 19 July and
30 Aug. Pollen samples
werecollected during this period but because of excessive levels of starch these samples were difficult
to filter during the
extraction process. H ERBERT
etal. (1985) identified
corn(Zea mays)
asthe predominant pollen source in Beltsville, MD. during this period. T ODD and
B
RETHERICK (1942) noted that while
mostpollens contain less than 3 % starch,
the levels in
cornpollen often exceeded 20 %.
DISCUSSION
It
wasof interest how the levels of total thiamine
asdetermined by chromatographic methods would compare with the levels determined by the
classical method (V IVINO and P ALMER , 1944). These authors reported the
average values of total vitamin B, in air dried pollen collected from four
periods during the season. Only total vitamin B, was reported since individual
reported since individual
forms of the vitamin
can notbe identified by this method. The agreement between the values for total thiamine by the
twodifferent methods is remark-
ably good with the possible exception of their (VmINO and P ALMER , 1944) early May collection which was lower than
ours. In the older methodologies for the analysis of vitamin B I , the phosphate forms
are enzymatically converted
to
thiamine and the total
amountof vitamin B 1 ,
notthe individual vitamers, is
then determined. With modern high performance liquid chromatography, the
various forms of vitamin B,
arefirst extracted from the sample, separated
on acolumn, and the
amountof each determined by the response of
afluorometric detector
asthe individual forms elute from the column. The total vitamin B, is then determined by addition of the vitamer concentrations.
Because of the large numbers of analyses that have
tobe performed in biological research, laboratory robotic systems
areincreasingly favored in vitamin research (H IGGS
etal., 1986). Robots
canbe programmed
toperform practically all of the manual operations performed in the laboratory from
extraction
toanalytical procedures. The
useof
arobotic system
tocompletely
automate the procedure from extraction
toanalysis should open
newhorizons in
areasof bee nutrition, toxicololy and physiology.
In summary, vitamin B, levels in bee pollen
asdetermined by the
automated liquid chromatographic method
arein good agreement with the
levels obtained by the classical method lending credence
tothe idea that both
procedures
arereliable. The chromatographic procedures does, however, yield
information
onthe different forms of the vitamin present which will be helpful
in future studies
onthe bio-availability of the different forms.
Received
for publication
in June 1986.Accepted for publication
inSeptember
1986.RÉSUMÉ
UTILISATION DE LA CHROMATOGRAPHIE LIQUIDE ET DE LA ROBOTIQUE POUR
DÉTERMINER
LES TENEURS EN THIAMINEET SES ESTERS DU POLLEN
RÉCOLTÉ
PAR LES ABEILLESLes teneurs en thiamine
(B I )
et ses formes mono etdiphosphate
ont été déterminées dans dupollen
frais
prélevé
dans des colonies d’abeilles enplein air,
àBeltsville, Maryland.
Le processusd’analyse
a étéentièrement éxécuté par le
système robotique
du laboratoire(Fig. 1) qui
se compose d’un brasqui peut bouger
dans les trois dimensions etqui possède
deux mainsinterchangeables
multifonctionnelles,capables
de
recouvrir,
demélanger,
de tourner,d’injecter,
et d’un modulequi
assure la communication entre lesystème robotique
et les autres instruments utilisés dans leprocédé d’analyse.
Les vitamines ont été extraites du
pollen
par unprocédé
semblable à celuiemployé
pour la vitamineB
6’
Dix ml à 5% (w/v)
d’acidesulfosalycilique
et 10 ml d’hexane ont étéajoutés
dans un tubeéchantillon contenant 1 g de
pollen
et un volume connu de standardinterne,
«amprolium
». Lafigure
2présente
unchromatogramme
type pour lesmélanges
de standards et pour un extrait depollen.
La
figure
3présente
les teneurs saisonnières en thiamine(w
moles/10g)
dupollen
récolté par les abeilles. Les trois formes de cette vitamine sontprésentes
dans les échantillons depollen
mais lathiamine est la forme la
plus
courante de lavitamine ;
vient ensuite lediphosphate,
suivi dumonophos- phate.
Les échantillons depollen
contenant laplus grande quantité
de vitamine sont ceux duprintemps (récoltes
du 12avril,
31 mai et du 7juin)
et ceux du début de l’automne(récolte
du 4octobre).
Lateneur
dépend
apparemment de la saison et del’origine
florale.ZUSAMMENFASSUNG
BESTIMMUNG DES THIAMIN-SPIEGELS UND SEINER ESTER IN BIENEN-GESAMMELTEM POLLEN
DURCH AUTOMATISIERTE
FLÜSSIG-CHROMATOGRAPHIE
Der Gehalt an Thiamin
(B 1 )
und seiner Mono- undDiphosphatverbindungen
wurde in frischem Pollenbestimmt,
der von freifliegenden
Völkern inBeltsville, Maryland, gesammelt
worden war. Der gesamteAnalysengang
wurde durch einLaboratoriums-Robotersystem ausgeführt (Fig. 1),
bestehend auseinem in drei Ebenen
beweglichen
Arm und zwei austauschbarenMehrzweckhänden,
die solche Vor-gänge
wieVerschließen,
Mischen, Drehen undInjizieren
ausführenkönnen,
und einemModul,
das die Kommunikation zwischen demRobotsystem
und den anderen in derAnalysenprozedur
benütztenInstrumenten herstellt.
Die Vitamine wurden aus dem
Bienenpollen
nach einem ähnlichen Verfahrenextrahiert,
wie es für VitaminB 6
benutzt wurde. Zehn ml von 5 %(w/v) Sulfosalizylsäure
und 10 ml Hexan wurden einemProbenglas zugesetzt,
das 1 g Pollen und eine bekannteMenge
eines internenStandard-Amproliums
enthielt. Ein
typisches Chromatogramm
einerMischung
aus Standard und einem Extrakt ausBienenpol-
len ist in
Fig.
2dargestellt.
Die saisonalen
Schwankungen
derThiaminmengen (w
mol/100g)
inbienengesammeltem
Pollen sind inFig.
3dargestellt.
Alle drei Formen dieses Vitamins sind in denPollenproben vorhanden,
aber Thiamin ist diewichtigste
Form des Vitamins. Diezweithäufigste
Form war dasDiphosphat, gefolgt
vondem
Monophosphat.
DiePollenproben
enthielten die höchstenVitaminmengen
imFrühjahr (Proben
vom12.
April,
31. Mai und 7.Juni)
und im Frühherbst(Probe
vom 4.Oktober).
DieMenge hängt
offensichtlich von der Jahreszeit und der Blütenart ab.
LITERATURE
H
AGEDORN H.H., BURGER M., 1968. - Effect of the age of
pollen
used inpollen supplements
on theirnutritive value for the
honeybee.
II. Effect of vitamin content onpollens.
J.Apic. Res., 7,
97-101.H
AYDAK
M.H.,
VrvtNOA.E.,
1943. -Change
in vitamin contentduring
the life of workerhoneybee.
Arch.
Biochem., 2,
201-207.H ERBERT
E.W.
Jr.,
SHIMANUKIH.,
1978. - Chemicalcomposition
and nutritive value of bee-collected and bee-storedpollen. Apidologie, 9,
33-40.H ERBERT
E.W.
Jr.,
VANDERSLICEJ.T.,
HIGGSD.T.,
1985. - Vitamin C enhancement of broodrearing by caged honey
bees fed achemically
defined diet. Arch. Insect Biochem.Physiol., 2,
29-37.H
IGGS
D.J.,
VANDERSLICEJ.T.,
HUANGM.H.,
1986. - Automated robotic extraction andsubsequent analysis
of vitamins in foodsamples.
In : Advances inlaboratory
automation - robotics 1985. Hawks, G.L. andStrimaitis,
J.(Eds.),
pp. 195-207.K
OCK
A.,
SCHWARZ1.,
1956. -Components
ofB-Complex
in the nutrition ofhoney
bees. lnsectesSociaux, 3,
213-228.L
UNDEN R., 1954. - A short introduction to the literature on
pollen chemistry.
Svensk Kem.Tidskr., 66,
201-213.T
ODD
F.E.,
BRETHERICK0.,
1942. - Thecomposition
ofpollens.
J. Econ.Entomol.,
35, 312-316.V
ANDERSLICE
J.T.,
MAIREC.E.,
DOHERTY R.F., BEECHERG.R.,
1980. -Sulfosalicylic
acid as anextraction agent for vitamin
B 6
in food. J.Agric.
Food Chem.,28,
1145.V
ANDERSLICE
J.T.,
HUANGM.H.A.,
1986. - Ahigh performance liquid chromatographic procedure
forthe
analysis
ofthiamine,
thiaminemonophosphate
and thiaminediphosphate
inbiological samples.
(In press).
V
IVINO