A sensitive and reproducible fluorescent-based HPLC assay for sphingomyelin synthase enzyme activity
They are equally contributed
A rapid, reproducible and accurate high-performance liquid chromatographic (HPLC) method for the quantitative determination of NBD-sphingomyelin in rat liver was developed and validated using reversed-phase silica gel column, methanol-water- trifluoroacetic acid (88:12:0.1 (v/v/v)) at a flow rate of 1 ml/min, isocratic elution, A fluorescence detector was used(λex = 475 nm,λem = 525 nm); 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl)-Sphingomyelin (NBD-SM) as an standard. Total run time was 16.0 min. The calibration curve was linear over the range of 0.0128-1.6 mg/l NBD-SM (R2 0.9997). The intra-day coefficient of variation ranged from 0.03 % to 1.8 %. The inter-day coefficient of variation over a period of 3 days ranged from 0.6 % to 4.3 %. The practical limit of detection was 0.006 mg/l with a quantification limit of 0.0128 mg/l. The HPLC method is found to be suitable for the quantitative estimation of sphingomyelin synthase -catalyzed reaction product and for studying inhibition mechanism of different inhibitors. The HPLC method has specific advantages over theTLC method.
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Keywords: HPLC; Sphingomyelin; Inhibitor; Rat;Liver ; Quantification
Sphingomyelin (SM), a phosphocholine ester of ceramide, is a principal structural and functional membrane component in mammalian tissue. SM is one of the major lipids in plasma lipoproteins and cell membranes. In clinical studies, SM plasma levels correlated with the occurrence of coronary heart disease independently of plasma cholesterol levels . SM and its derivatives are accumulated in human and experimental atherosclerotic lesions . However, sphingomyelin synthase (SMS) is the last enzyme involved in the SM biosynthesis that transfers the phosphorylcholine moiety from phosphatidylcholine (PC) onto the primary hydroxyl of ceramide producing SM and diacylglycerol (DAG) . It was reported that inhibition of SM synthesis was associated with a significant reduction in atherosclerotic lesion formation in apolipoprotein E- knockout mice. [4-6]. In a human case-control study, it was found that both plasma SM and SM/PC ratio are independent risk factors for coronary heart disease . These data suggest that SM might play a promoting role, where as PC might play a preventive role, in the development of atherosclerosis, and the measurement of their levels might provide new insights into atherogenesis in humans and in various mouse models as well. Consequently, most investigators have studied changes in SM , PC and ceramide levels in biological samples using chromatography with a variety of detection methods, such as thin-layer chromatography[8-11], ultra-violet (UV) detection[12-15], mass spectrometry (MS) [11,16-18] and evaporative light scattering detector (ELSD) [19,20]. Among these detection methods, their detection are generally most sensitive to phospholipids, but many studies have indicated that SMS is located mainly in the cis-, medial-Golgi  and trans-Golgi network , and plasma membranes . Therefore, SM presence is extensive and complex, there is a need for a rapid, specific, sensitive and inexpensive method to quantitate SM in the organism on a routine basis. In this article, we describe what we believe is such a method. The specificity of the method was demonstrated by comparing retention time and fluorescent spectra of the NBD-Sphingomyelin peak obtained in the enzyme-catalyzed reaction mixture with the NBD-Sphingomyelin standard which does not exist endogenously in the organism.
2.1. Chemicals and reagents
C6-NBD ceramide and C6-NBD sphingomyelin from bovine brain were obtained from Matreya, Inc. (Pleasant Gap, PA).Methanol (HPLC grade) and trifluoroacetic acid (HPLC grade) were also purchased from Sigma Chemical Co. Laboratory-prepared water (Milli-Q) was used throughout the study. Other
chemicals, if manufacturer is not mentioned, were obtained from Sigma Chemical Co. (St. Louis, MO).
The HPLC system was equipped with Agilent 1100 Series system (Agilent Technology, Palo Alto, CA, USA) consisting of vacuum degasser, column thermostat, quaternary pump, fluorescence detector (FLD) and a Chemstation computer software system. Chromatographic analysis was carried out using a Zorbax Rx-SIL silica gel (25 cm-4.6mm i.d., 5μm particle size, 80 Å pore size) column from Rockland Technologies, Inc., USA.
BASE III image analyzer system (Fuji,Tokyo, Japan).ç”Ÿç‰©ç”µæ³³å›¾åƒåˆ†æžç³»ç»Ÿ å¤-FR-200
2.3. Rat liver tissue preparation (Sample preparation)
Sprague-Dawley rats (200-300 g) were used. These were maintained in restricted-access rooms with a controlled temperature(23 â„ƒ) and a 12 h light-dark cycle and were allowed free access to standard laboratory diet and tap water. All experimental procedures were approved by the Institutional Review Board and all attempts were made to reduce pain and discomfort to experimental animals. Animals were sacrificed and liver was dissected, weighed and then homogenized with 0.8 ml of SHT buffer (0.25M Sucrose, 10mM Hepes, 50mM Tris, pH 7.4). The supernatant was used for both the TLC as well as the HPLC assays.
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2.4. TLC analysis of sphingomyelin synthase activity
The SM synthase activity was detected according to the method of C. Yeang et al [9,24,25]. Reaction mixtures contained 50 mM Tris-HCl (pH 7.4), 25 mM KCl, 4 μL C6-NBD-ceramide (0.1 μg/μl), phosphatidylcholine (0.01 μg/μl), homogenate suspension. Incubation was performed at 37 â„ƒ for 120 min . The reaction was stopped by adding 2 ml chloroform/methanol (2:1) and vortexed and centrifuged. Lower phase was collected,and solvent was evaporated, dried under N2 gas, resolved using the solvent system.
Aliquots were applied to TLC plates, and separated by thinlayer chromatography using Chloroform:
2.5. HPLC method for the analysis of sphingomyelin synthase activity
An isocratic solution was used to separate the substrate and product of the enzyme reaction. The isocratic mobile phase containing methanol-water-trifluoroacetic acid (88:12:0.1, v/v/v) was used. The column temperature was maintained at 25â„ƒ. Mobile phase flow rate was set to 1.0 ml/min with a run time of 16 min. The FLD was operated (excitation 475 nm, emission 525 nm) with the recorder response set at 0.4 absorbance units per full scale deflection (AUFS). The injection volume was set at 20μl. The samples were quantified using linear regression of response NBD-SM peak area versus NBD-SM concentrations. Data was collected and processed using a Chemstation software for HPLC system.
Kinetic measurements for sphingomyelin synthase were carried out using different concentrations of SMS homogenate upto 500M in 20mM of assay buffer (pH 7.4) in a final volume of 1ml assay mixture. The reaction was started with the addition of 0.00275 units of sphingomyelin synthase and was terminated
by adding chloroform/methanol (2:1) to a final concentration of 0.3 M. Denatured proteins were precipitated by centrifugation at 5000 rpm for 10 min .Clear supernatants were processed for HPLC experiments.
The mobile phase solvents (methanol ,water and trifluoroacetic acid ) were filtered through 0.22μm nylon membrane and further degassed before use.
2.6. Method validation for HPLC method
2.6.1. Linearity and range
Standard stock solutions of NBD-SM (1 mg/ml) was prepared by using ethanol . Successive dilutions of NBD-SM standards were made to obtain the seven levels of calibration standards having the following concentrations:1.6, 0.8, 0.32, 0.16 ,0.064, 0.032 and 0.0128 mg/l. All the resulting working solutions were kept in the refrigerator (4â„ƒ) during manipulation. The solutions were injected in duplicate for obtaining a representative result.
Injections of three different concentrations (0.8, 0.2 and 0.08 mg/l), each in triplicate were injected on the same day and the relative standard deviation was calculated to determine the precision of intra-day and inter-day variation of our experimental result.
Percentage recovery was calculated from the difference between the calculated and expected concentration (calculated from the corresponding peak area). The recovery studies were carried on with four concentrations of the product (25,50, 100 and 200_M NBD-SM ) to find out the accuracy of the
3.1.TLC analysis of sphingomyelin synthase activity
The use of fluorescent NBD-ceramide as the substrate for measuring SM synthase activity obviates the need for producing radiolabeled PC, but this method need a fluorescence detector .To use NBD-ceramide as the substrate, resuspend 6 nmol NBD-ceramide (dried from an ethanol stock) in 40μL of 1% fatty acid-free bovine serum albumin (BSA). Add 60 μL SM synthase reaction buffer and incubate at 37°C for 120 min before use.NBD-ceramide and NBD-SM were visualize by UV irradiation and measured by a TLC scanner with fluorometer (excitation 475 nm; emission 525 nm). To calculate NBD-SM content, the positive spots on TLC plates were measured using the BASE III image analyzer system(Fig.1).
3.2. Development of HPLC method for the kinetic analysis of SMS activity
In order to eliminate the TLC related difficulty we have used the HPLC method as a simple and efficient alternative method for the purpose of assaying the same reactions catalyzed by sphingomyelin synthase. The aim of this work was the development of a rapid HPLC assay with a total run time 16 min, while achieving suitable sensitivity and selectivity. The mobile phase was chosen after several trials with methanol and water solutions in various proportions and at different pH values. A mobile phase consisting of methanol-water-trifluoroacetic acid (88:12:0.1, v/v/v) was selected to achieve maximum separation and interference-free chromatogram. Changing the mobile phase composition to 88% methanol led to a better run time. Flow-rates between 0.5 and 2 ml/min were studied.A flow rate of 1.0 ml/min gave an optimal signal to noise ratio with a reasonable separation time. The selected chromatographic conditions provided good resolution of NBD-SM and the NBD- ceramide.Identification of phospholipids was carried out by comparison with the retention time of pure standards. The average retention times for NBD- SM and NBD-ceramide were observed to be 13.673, 14.817 min respectively (Fig. 2).Typical chromatogram
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of NBD- SM and NBD-ceramide is shown in (Fig. 2).The sample was obtained from liver of adult rat with 1 mg/ml of NBD-ceramide added before extraction. The chromatogram shows that NBD- SM and NBD-ceramide are separated completely and none of the liver endogenous components interfere with the assay (Fig. 3). Inhibitor YZDS and PC were not found the corresponding peaks in the chromatogram(Fig. 4).Fig. 5 shows the time dependent enzymatic reaction, where we observed a gradual increase in the area under the peak of NBD-SM. Using this HPLC method we further calculated the initial rates of the sphingomyelin synthase reaction.
The effect of the standard inhibitor, YZDS were also studied and the sphingomyelin synthase reaction kinetics was followed using the HPLC method. YZDS exhibited a clear dose dependent inhibition (Fig. 6).
Hence data obtained from HPLC method gives a true representation of the enzymatic rate and inhibition mechanism and can be easily extended to calculate the IC50 and the inhibition constant of an inhibitor or drug.
3.3. HPLC method validation
3.3.1. Linearity and range
With the methanol:water:trifluoroacetic acid mobile phase, the response of the product (NBD-SM) was linear in the concentration range between 0.0128 and 1.6 mg/l. The mean correlation coefficient was calculated to be 0.9997 . the corresponding regression equation was y = 139403x - 0.0516, where y is the NBD-SM peak area ratio and x is the NBD-SM concentration (mg/l). The linear curves in this range of
concentration and the correlations make it suitable for quantitation.
The recovery percentage was calculated for triplicate sets of 25, 50, 100 and 200 M concentrations of the product (NBD-SM) and were found to be 100.7, 107.1, 101.7 and 100.3%,respectively.
Extraction yield assays led to mean absolute recovery of NBD-SM to be 90.69%. All the values obtained were within the limits required for biological samples, 100±15% for absolute recovery (
the analyte ) and less than 15% for CV .
The reproducibility and precision of the data were evaluated through intra-day and inter-day studies, for the product concentrations of 25, 50 and 100 M. Concentrations measured for the intra-day concentrations (±S.D.) werefound to be 26.783±0.852_M, 49.286±0.195_M and 99.007±0.348 whereas the inter-day concentrations were calculated as 27.563±1.12_M, 51.693±2.105_M and 105.236±5.053, respectively. As is evident from the resolution of the peaks, it is well evident that this process is sufficiently specific for the separation of NBD-SM and the NBD- ceramide.
The specificity of the method was demonstrated by comparing retention time in the lipid extract with then NBD-Sphingomyelin standard.No chromatographic interferences were found with tested molecules such as NBD- SM and NBD-ceramide .When biological sample was treated with specific sphingomyelinase synthase, all of the fluorescence compounds in the NBD-SM region of the chromatogram disappeared as shown in Fig. 2. Fig. 2 showed only two peaks within the run time, specifically the NBD- SM and NBD-ceramide. This demonstrates that the method is specific for NBD-SM and no other fluorescence contaminant co-eluted with the NBD-SM peak.
3.3.5. Limit of detection and quantification
The limit of detection and the limit of quantitation experimentally verified by six injections ofNBD-SM at the limit of detection and the limit of quantitation concentrations,were 0.0064 mg/l (6.839M) and 0.0128 mg/l (13.679 M) respectively. Since endogenous SM concentrations in rat liver, have been reported in the millimolar range(aproximately 0.56 mM), our method is validated to be sufficiently sensitive to detect endogenous SM concentrations.
3.4. Extension of the HPLC-based to cell line lysate
Assays for the presence of the enzyme, substrate and
the product using spectrophotometric method with cell
line or tissue lysate present more problems of non-specific
contribution. Therefore we have analyzed the time dependent
product formation profile in the Hut-78 (T cell lymphoma)
cell line lysates using HPLC-based method and have
been able to monitor the substrate utilization kinetics
TLC method is popularly used for the estimation of sphingomyelinase synthase activity but it suffers from the resolution problem of the substrate, product and inhibitor. The main drawback with the TLC
method lies in the time required for the preparation of samples. Moreover, higher concentration of enzyme is also required in the TLC method to give a quantitative estimation of the product formation or substrate degradation and to check the effect of the inhibitor. SMS inhibitors are used as drugs and hence an exact dose is needed to be prescribed, therefore any technical problem in calculating the true dose is not at all desirable.Thus the alternative HPLC method is suitable for the quantitative measurement of the kinetics and inhibition of mammalian SMS. The method can also be used for the screening of a large number of inhibitors of SMS. HPLC method for the assay of SMS activity or inhibition was proved to be quantitative,
reliable, accurate and cheap.
We have developed a new HPLC method for quantitatively studying the kinetics and inhibition of mammalian SMS using enzyme system or using cell extracts and body fluids. The same method can also be extended for screening a large number of compounds for the discovery of SMS inhibitors.