2İnönü University, Faculty of Medicine, Department of Medical Biochemistry, Malatya, Turkey
3İnönü University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Malatya, Turkey
4İnönü University, Faculty of Pharmacy, Department of Analytical Chemistry, Malatya, Turkey
5Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Ankara, Turkey DOI : 10.12991/201216399
Summary
Kolesterol molekülünün doğada sadece bir stereoizomerik formda, nat-kolesterol, bulunduğu bilinmektedir. Nat-kolesterol ve enantiomeri olan ent-kolesterol, biyolojik moleküllerle bazen enantiospesifik etkileşimler göstermektedir. Bu durumda, kolesterol doğada bir formda bulunuyorsa “kolesterol neden enantiomerik seçicilik göstermektedir? ” sorusu ortaya çıkmaktadır. Bu amaçla, domuz karaciğeri ve koyun yün yağından elde edilen kolesterolün stereokimyasal analizleri, ters-faz, mobil faz modifiye edici ajan olarak farklı siklodekstrinlerin kullanıldığı ters-faz ve kiral' den oluşan üç farklı HPLC metodu ile gerçekleştirilmiştir. Her iki kolesterol örneğinin, permetillenmiş γ-siklodekstrin ve amiloz tris-(3,5-dimetilfenilkarbamat) kiral kolonlar ile yapılan HPLC analizlerinde kolesterol stereoizomeri bulunmamaktadır. Bununla birlikte, domuz karaciğerinden elde edilen kolesterol örneğinin çeşitli siklodekstrinlerin mobil faz modifiye edici olarak kullanıldığı ters-faz HPLC analizlerinde belirlenen bir pik, koyun yün yağından elde edilen kolesterol örneklerinin analizinde gözlenmemektedir. Öte yandan, farklı bekletme koşullarındaki kolesterol örneklerinin ters-faz HPLC analizleri ile yeni hazırlanmış kolesterol örneklerinin siklodekstrinlerin mobil faza eklendiği ters-faz HPLC analizleri sonucunda, kromatogramlarda hemen hemen birbirine eş değişiklikler gözlenmektedir. Bu durum siklodekstrinlerin katalitik özelliklerine dayandırılabilir. Bu nedenle, kolesterolün HPLC ile stereoizomer analizinde mobil faz modifiye edici olarak siklodekstrinlerin kullanılması uygun olmayabilir.Introduction
Cholesterol plays important roles in many functions of cells. Presence of cholesterol in cells in right amount and place is necessary for membrane structure and function, signal transduction and as a result, for human health[1]. Additionally, cholesterol metabolites e.g. steroids and bile acids have important biological roles including signal transduction as well as lipid solubilization[2]. On the other hand, unbalanced cholesterol distribution has been found to be related to many disorders including atherosclerosis, coronary artery disease, hypertension, Alzheimer, diabetes as well as cancer[1].Cholesterol has tetracyclic cyclopentanoperhydrophenanthrene skeleton with a hydroxyl group attached to carbon 3 and a side chain at carbon 17. Cholesterol consists of polar head group and apolar hydrocarbon body conferring the molecule amphipathic property. Its fused tetracyclic structure and eight chiral centers make 256 stereoisomers of cholesterol molecule possible[3].
It has been reported that cholesterol occurs naturally in only one stereoisomeric form, i.e. natcholesterol, owing to the nature of enzymatic reactions of steroid synthesis[1]. However, it is known that enantiospecific cellular proteins take part in cholesterol absorption in hamsters[4]. Additionally, ent-cholesterol, enantiomer of cholesterol, and nat-cholesterol exhibit enantiomeric interaction with cholesterol oxidase but not with epidermal growth factor[1]. Then the question arises “if cholesterol naturally occurs in only one form, why interaction with cholesterol exhibit an enantiomeric selectivity? To this end, stereoisomeric homogenity of cholesterol from porcine liver and wool wax has been investigated using HPLC method with C18 column with and without cyclodextrins as mobile phase modifier and two different chiral columns.
Analysis with permethylated γ-cyclodextrin (γ-CD), and amylose tris-(3,5-dimethylphenylcarbamate) (ADMPC) chiral columns showed that no cholesterol stereoisomer is present. However, HPLC analysis using C18 column with cyclodextrins as mobile phase modifier, a peak was detected in cholesterol from porcine liver which was not found in cholesterol from wool wax.
EXPERIMENTAL
Materials
All chemicals and solvents were of analytical grade. Cholesterol
obtained from porcine liver (C-PL) was purchased from Sigma
(Saint Louis, Missouri, USA) and cholesterol obtained from
wool wax (C-WW) from Acros Organics (NewJersey, USA).
HPLC grade isopropanol from Merck KGaA (Darmstadt,
Germany) and acetonitrile were purchased from JT Baker
(Deventer, Holland). Cyclodextrins; α-cyclodextrin,
β-cyclodextrin, β-methylcyclodextrin,
β-hydroxypropylcyclodextrin, γ-cyclodextrin,
γ-hydroxypropylcyclodextrin were purchased from Wacker-
Chemie AG (Burghausen, Germany). Ultrapure water used for
mobile phase was generated by water purification system
from Human Corporation (Seoul, Korea).
Instrumentation
An Agilent 1100 System (Agilent Technologies, Palo Alto, CA,
USA) comprising quaternary solvent delivery system, an on-line
degasser, a manual injection, a column temperature controller
and DAD detector was used for the analysis. Separations were
carried out using C18 column, 250x4,6 mm, 5 μm particle size
(ACE, Aberdeen, Scotland), permethylated gamma-cyclodextrin
(γ-CD), 200x4,6 mm, 5 μm particle size or, amylose tris-(3,5-dimethylphenylcarbamate)
(ADMPC) chiral columns, 250x4,6 mm,
5 μm particle size (Macherey Nagel, Düren, Germany).
Reversed-phase HPLC conditions
Chromatographic separations with C18 column were carried
out by isocratic system with mobile phase consisting of
isopropanol:acetonitrile:water (60:30:10, v:v:v). The mobile
phase was prepared daily and filtered through 0,45 μm
Milipore membrane and degassed for 15 minute in an ultrasonic
bath prior to use. Mobile phase was delivered to the system
at a flow rate of 1 mL/min and the UV detector was set at
210 nm[5].
Reversed-phase HPLC conditions with cyclodextrin as a
chiral agent in mobile phase
All the chromatographic parameters were the same as described
in reversed-phase HPLC conditions. 0.01% various cyclodextrins
(w/v) was added to mobile phase as a chiral agent.
Chiral HPLC conditions
Chromatographic separations with both chiral columns were
carried out by isocratic system. Mobile phase composition was
isopropanol:acetonitrile:water (60:30:10, v:v:v) for γ-CD column[5] whereas hexane:isopropanol (90:10, v:v) was used for
ADMPC column[6]. Mobile phase was delivered through the
system at a flow rate of 1 mL/min and the UV detector was set
at 210 nm for both columns. Mobile phase preparation was the
same as described in reversed-phase HPLC.
Sample Preparations
C-PL and C-WW were dissolved in mobil phase of HPLC column
used at a concentration of 200 ppm.
In order to investigate and compare the effects of storage conditions on C-PL and C-WW, freshly prepared samples were divided into three aliquots and subjected to either UV or different storage temperatures.
C-PL and C-WW samples dissolved in mobil phase at a concentration of 200 ppm were exposed to UV light in an open petri dish at room temperature for 3 hours. An aliquot of C-PL or C-WW samples were kept in a capped volumetric flask at room temperature for one month. Remaining aliquots were kept in a capped volumetric flask at +4 °C for 1 month.
Results
Investigation of cholesterol stereoisomers with chiral columnsChromatograms from analysis of freshly prepared samples of C-PL and C-WW by γ-CD chiral column were shown in Figure 1a and 1b. Results from both samples show that cholesterol peaks had same retention time and there was no different stereoisomer present. Void volume of γ-CD chiral column was calculated as 1.76 mL, and small peaks eluted in void volume were considered to be impurities. Considering the retention time of cholesterol peak it can be seen that cholesterol exhibits a poor interaction with γ-CD.
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FIGURE 1: Chromatograms obtained with γ-CD chiral column from analysis of freshly prepared samples of C-PL (a) and C-WW (b). |
When the stereoisomer homogenities of C-PL and C-WW were investigated with ADMPC chiral column, similar results were obtained. Retention time of cholesterol peaks for both samples and impurity were identical. Peaks eluted in void volume were also similar (Figure 2a and 2b).
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FIGURE 2: Chromatograms obtained with ADMPC chiral column from analysis of freshly prepared samples of C-PL (a), C-WW (b). |
Investigation of cholesterol stereoisomers with
cyclodextrins as mobile phase modifier agents
Various cyclodextrins were added to mobile phase of reversedphase
HPLC with C-18 column. To this end, 0.01% (w/v)
α-cyclodextrin, β-cyclodextrin, β-methylcyclodextrin,
β-hydroxypropylcyclodextrin, γ-cyclodextrin or
γ-hydroxypropyl cyclodextrin were added to mobile phase for
the analysis of C-PL and C-WW samples with reversed-phase
HPLC. Among cyclodextrins only γ-hydroxypropyl cyclodextrin
results were shown in Figure 3 and 4.
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FIGURE 3: Chromatograms of C-PL samples by reversed-phase HPLC analysis (a) without, (b) with the addition of γ- hydroxypropyl cyclodextrin. |
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FIGURE 4: Chromatograms of C-WW sample by reversed-phase HPLC analysis (a) without, (b) with the addition of γ- hydroxypropyl cyclodextrin. |
Retention times (RT) of cholesterol peaks (peak 2) varied between 15,612-17,599 depends on type of cyclodextrin added as mobile phase modifier. For C-PL sample, peak 1 (RT 10,846- 12,796) with shorter retention time than peak 2, and peak 3 (RT 18,312-21,547) with longer retention time than peak 2 were determined, but somewhat peak 3 was not determined in the analysis with γ-cyclodextrin. For C-WW sample, unlike C-PL sample, peak 1 was determined by reversed-phase HPLC both with and without cyclodextrin addition as mobile phase modifier (Figure 4) (chromatograms of other cyclodextrins were not shown).
Reversed-phase HPLC analysis carried out by adding cyclodextrin to mobile phase for C-PL and C-WW, retention times of peaks 2 and peaks 3 overlap for all types of cyclodextrins. However, retention time of peak 1 was calculated as 11,44±0,5 for C-PL sample, whereas 12,12±0,41 for C-WW sample for 6 different cyclodextrins. Elution time of peak 1 for C-PL sample was 0,69±0,13 min. shorter than C-WW sample and peak height was found to be approximately two times higher than that of C-WW sample.
According to results of reversed-phase HPLC analysis, number of peaks related with impurities between 2,3-3,5 min. retention time increased by adding cyclodextrins to mobile phase. Initial number of impurity peaks between 1,5-4,2 retention times was 3 or 4, whereas after addition of cyclodextrins to mobile phase number of peaks increased to 8 or 9 (Figure 3 and 4). In order to define these peaks whether probable stereoisomer or decomposition products of cholesterol, reversedphase HPLC analysis of C-PL and C-WW samples kept under different storage conditions were conducted.
Investigation of stereoisomers of samples kept under
different storage conditions
C-PL ve C-WW samples were dissolved in mobile phase, kept
under UV light or room temperature, or at +4 °C, and reversedphase
HPLC chromatogram of these samples were compared
with that of freshly prepared samples (Figure 5 and 6).
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FIGURE 5: Chromatograms of reversed-phase HPLC analysis of C-PL samples under different conditions. Freshly prepared (a), exposed to UV light (b), kept at room temperature (c), kept at +4 °C (d). |
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FIGURE 6: Chromatograms of reversed-phase HPLC analysis of C-WW samples under different conditions. Freshly prepared (a), exposed to UV light (b), kept at room temperature (c), kept at +4 °C (d). |
For both cholesterols analysis, new small peaks with the retention time ranging between 1,6 to 4,4 and peak 3 appeared in samples kept at room temperature and +4 °C for a month. Although peak 1 was present in C-WW sample, this peak also appeared after exposition to UV light. Peak 2 has the same retention time for both cholesterol samples. As a result, chromatograms of reversedphase HPLC analysis of samples kept at room temperature and at +4 °C bear resemblance with chromatograms of reversed-phase HPLC analysis of freshly prepared cholesterol samples carried out by adding cyclodextrins to mobile phase. Retention times of peak 1 and peak 3 overlapped with retention times of peak 1 and peak 3 of analysis with cyclodextrins of all types.
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