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 » Introduction
 »  Materials and Me...
 » Results
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RESEARCH ARTICLE
Year : 2022  |  Volume : 54  |  Issue : 4  |  Page : 270-277
 

Simultaneous determination of lactulose, sucrose, sucralose, and mannitol using high-performance liquid chromatography-refractive index to estimate intestinal permeability in patients with active ulcerative colitis


1 Department of Pharmacology, Post Graduate Institute of Medical Education and Research, Chandigarh; Department of Biochemistry, Pt. Jawahar Lal Nehru Government Medical College, Chamba, Himachal Pradesh, India
2 Department of Gastroenterology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
3 Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, Mohali, Punjab, India
4 Department of Pharmacology, Post Graduate Institute of Medical Education and Research, Chandigarh, India

Date of Submission03-Feb-2022
Date of Decision24-Aug-2022
Date of Acceptance26-Aug-2022
Date of Web Publication04-Oct-2022

Correspondence Address:
Dr. Bikash Medhi
Department of Pharmacology, Post Graduate Institute of Medical Education and Research, Chandigarh - 160 012
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijp.ijp_90_22

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 » Abstract 


OBJECTIVES: The intestinal permeability (IP) of sugars and their derivatives has been widely used to assess mucosal damage in gastrointestinal diseases. Ulcerative colitis (UC) is a recurring and relapsing disease that causes inflammation of the gut. IP of sugars can be evaluated and correlated with the flare of UC.
MATERIALS AND METHODS: A prospective study was conducted on 91 patients with active UC at the tertiary care center in North India. Mayo grading system assessed disease activity, and IP was assessed by measuring sucrose, lactulose, mannitol, and sucralose in urine samples from UC patients. A high-performance liquid chromatography (HPLC) method to detect all of these sugars simultaneously using a refractive index detector was developed and further validated in patients with UC.
RESULTS: The analytical recovery rate of the tested sugars ranged from 95% to 146% in the urine matrix. The limit of detection and limit of quantification were 78.838 mg/L and 262.79 mg/L for sucrose, 84.994 mg/L and 283.31 mg/L for lactulose, 74.789 mg/L and 249.30 mg/L for mannitol, and 50.908 mg/L and 169.69 mg/L for sucralose.
CONCLUSION: The standardized HPLC method is sensitive and suitable for the simultaneous detection and determination of different sugar moieties in the urine sample. Patients with UC can be evaluated indirectly for the flare by estimating the recovery rate of sugars through gut permeability. The procedure is noninvasive and thus improves the quality of life of chronically ill patients.


Keywords: High-performance liquid chromatography, intestinal permeability, ulcerative colitis


How to cite this article:
Sarotra P, Dutta U, Gupta H, Ravindranathan Kartha K P, Kochhar R, Prakash A, Sarma P, Shah J, Medhi B. Simultaneous determination of lactulose, sucrose, sucralose, and mannitol using high-performance liquid chromatography-refractive index to estimate intestinal permeability in patients with active ulcerative colitis. Indian J Pharmacol 2022;54:270-7

How to cite this URL:
Sarotra P, Dutta U, Gupta H, Ravindranathan Kartha K P, Kochhar R, Prakash A, Sarma P, Shah J, Medhi B. Simultaneous determination of lactulose, sucrose, sucralose, and mannitol using high-performance liquid chromatography-refractive index to estimate intestinal permeability in patients with active ulcerative colitis. Indian J Pharmacol [serial online] 2022 [cited 2022 Dec 3];54:270-7. Available from: https://www.ijp-online.com/text.asp?2022/54/4/270/357833





 » Introduction Top


Intestinal permeability (IP) of the nonmetabolizing urinary sugars has been broadly utilized as a noninvasive technique to assess gut mucosal integrity. The nonmetabolizing sugars in urine, such as lactulose, sucrose, mannitol, and sucralose, are site-specific probes and are passively absorbed along the gut.

Sucrose is generally absorbed in the gastric mucosa and then metabolized by the enzyme sucrase and has been widely used to assess the gastroduodenal mucosal integrity.[1] Mannitol is smaller in size and therefore easily transverses the villous tips made up of tight junction proteins. It has also been considered one of the markers of epithelial injury. Lactulose, a large-sized disaccharide, can also transverse the junction pores in the villous base and along the crypt. The permeation of lactulose depends on the leakiness via pores.[2] Sucralose is impervious to the action of sucrase. It is also passively absorbed along the pores of the intestines before being excreted in the urine. Therefore, it has also been used to assess colonic permeability.[3] The permeability of the gastrointestinal (GI) mucosa was generally delineated by expressing the percentage recovery ratios of sugars. Elevated sucralose/mannitol ratios (SMR) determine overall GI permeability, while the elevated lactulose/mannitol ratio (LMR) indicates small intestinal damage.

The assessment of the aforesaid IP is widely used in clinical practice. Patients with various pathologies such as inflammatory bowel disease, celiac disease, various acute and chronic diarrheal diseases, and type I diabetes were examined for leakiness in the GUT by IP of different sugars.[4],[5],[6],[7] The studies also portray sugar moieties as ideal compounds for assessing mucosal damage by measuring the IP of sugars in patients with ulcerative colitis (UC).

UC is a chronic relapsing-remitting GI illness with recurrent inflammation of the colonic mucosa that affects millions worldwide. In India, the prevalence of UC is highest in the Punjabi population, with a rate of 6.02 cases per 100 persons.[8] Various clinical and endoscopic scoring systems (e.g., Mayo score) have been used in clinical trials to prove treatment efficacy and disease remission.[9] Endoscopic scoring systems are the gold standards for assessing disease. However, chronic relapsing disease causes discomfort and pain and affects patients' quality of life. Therefore, determining IP of sugars can correlate with disease indices and help assess damaged mucosa.

Various methods using enzymatic, calorimetric, thin-layer chromatographic, and gas chromatographic techniques have been widely used to determine the concentration of intestinal sugars.[10] Several gas chromatography (GC) and high-performance liquid chromatography (HPLC) methods derivatize and analyze sugars using gas chromatography with flame ionisation detection or gas chromatography mass spectrometry and direct high-performance anion-exchange chromatography with pulsed amperometric detection method.[11],[12] However, these methods are often tedious, require derivatization of sugars, expensive reagents, are cumbersome, time-consuming and require expertise. In addition, most of these methods do not allow simultaneous determination of the sugar concentrations, possibly affecting the accuracy of end results. Although the latest method, evaporative light scattering detector (ELSD), has shown high sensitivity to detect these sugars, it also has limitations such as the use of a nebulizer assembly, which increases operational costs, sample destruction, and the calibration produced is often nonlinear.[13] In the present study, the simultaneous assessment of four sugars gives a better picture of the total IP across different parts of the GI tract. None of the studies assess urinary excretion of four sugars simultaneously, adding novelty to our methodology. Searching PubMed using the keywords (Intestinal permeability AND Urine AND sugar AND HPLC) yielded 57 results, of which only 47 studies were clinical. Most of these studies used the simultaneous detection of two sugars in urine, with lactulose and mannitol being the most common.[14] Few studies have used the simultaneous detection of three sugars in human urine, e.g., lactose, mannitol, and sucrose or rhamnose, mannitol, and lactulose or mannitol, lactulose, and glucose using HPLC MS-MS-based method.[15],[16],[17] Only two studies investigated simultaneous analysis (rhamnose, lactulose, 3-O-methyl-D-glucose, and xylose) using HPLC with anion exchange columns or fluorescent labeling.[12],[18] The present study was conducted to evaluate the performance of the simultaneous detection of four different sugars in urine (lactulose, sucrose, sucralose, and mannitol). The validation was performed on clinical samples from IBD patients (the leaky gut in IBD is well known) using a relatively easy-to-use HPLC method, highlighting the novelty of the present study.

Therefore, the present research investigation was designed to standardize a technique that is rapid, highly sensitive, and designed for the simultaneous analysis of four sugars (urine recovery samples) using HPLC equipped with a universal refractive index (RI) detector and deionized water (a nontoxic) as a solvent system. Therefore, a clinical study was designed to evaluate IP in UC patients and its correlation with disease characteristics.


 » Materials and Methods Top


The clinical trial was approved by the Institutional Ethics Committee PGIMER (PGI/IEC/2012/1247-48, dated September 03, 2012) and conducted in accordance with the of the World Medical Association's Code of Ethics for Experimentation on Humans (Declaration of Helsinki). The trial is registered under the number CTRI/2011/08/001944 in the Clinical Trials Registry. Patients aged over 18 years and under 65 years with active UC attending PGIMER Gastroenterology OPD, Chandigarh, were enrolled in the study after obtaining written informed consent. UC was diagnosed based on clinical manifestations. The number of stools per day with mucus, blood, and tenesmus (UC disease activity index scoring system) and endoscopic features (vascular pattern, friability, ulceration, and pseudopolyps) were observed for the disease activity. The patients were treated with standard therapy consisting of mesalamine, steroids, and azathioprine.

Lactulose, D-mannitol, sucrose, and calcium nitrate were purchased from Sigma Chemical Co. Sucralose was obtained from JK Chemicals Pvt. Ltd. Syringe filters (Nylon 66, 0.2 μm membrane filters) were procured from Advanced Microdevices. Durapore PVDF 0.4 μm membrane filters were purchased from Millipore. Deionized (DI) water, 18MΩ-cm resistivity (ELGA RO purification system) was used as the eluent for HPLC analysis. Bond Elut NH2 500 mg, 3 mL cartridges from Agilent Technologies were used in the sample preparation.

The permeation of the unmetabolized sugars such as sucrose, lactulose, mannitol, and sucralose into the intestinal mucosa can be measured by IP tests. Subjects were told to eat light, digestible food, nondairy food, and fasted overnight. Gut permeability was assessed in the subjects by administering a solution containing 100 g sucrose, 5 g lactulose, 2 g mannitol, and 2 g sucralose in 500 mL of water. Urine was collected after 5–6 h. The subjects who have passed the urine before 5 h were not included in the study. The total volume was recorded. The urine sample was vigorously shaken and aliquots were frozen at −80°C for subsequent analysis. The ratio of the percentage excretion of the ingested dose of sucrose and lactulose indicates gastroduodenal permeability; lactulose and mannitol indicate small IP; sucralose and mannitol indicate colonic permeability.[19]

The analysis was performed on a Waters 600 series instrument equipped with a Delta 600 Quaternary Pump, a 600 Controller, 2414 Refractive Index Detector, and an Advanced Laser Polarimeter Detector system (PDR-Chiral Inc., USA). Waters Empower Chromatography Software was used for analysis.

Stock solutions of sucrose (7.10, 3.55, 2.10, 1.05, and 0.525 mg/L), mannitol (12.20, 8.90, 6.10, 4.45, 2.225, and 0.556 mg/L), lactulose (12.10, 6.05, 4.10, 2.05, 1.025, and 0.512 mg/L), and sucralose (20.10, 15.90, 10.05, 7.95, 3.975, and 0.993 mg/L) were prepared in the urine of a nondiseased subject (control). The standards were each vortexed for 10 s. Aliquots (500 μl) were filtered through syringe filters (Nylon 66, 0.2 μm), and 20 μl was used to inject into the HPLC. The limit of detection (LOD) and limit of quantification (LOQ) were determined from this standard analysis. The mean run-to-run and day-to-day variability was assessed by using the coefficient of variation (C.V.) obtained by dividing the standard deviation by the mean values for the concentrations (C.V. = SD/mean). Agilent Technologies Bond Elut Amino 500 mg cartridge was conditioned with 5 mL of methanol, followed by 5 mL of methanol and water. A urine sample (3 mL) was filtered through the cartridges and initially 1 mL of urine was discarded. The residual volume collected was gas dried by slowly blowing the air under pressure. HPLC grade deionized water (1 mL) was added to the samples and filtered through syringe filters (Nylon 66, 0.2 μm). Final aliquots were analyzed by HPLC. Analysis of the sugar sample probe was performed using Waters calcium-bonded sugar Pak-I column (300 mm × 6.5 mm [ID], 10-μm particle size). Chromatographic separation was achieved using HPLC-grade water (18 Ω) as the mobile phase (25 min at a flow rate of 0.5 mL/min). The eluent was filtered through a 0.45 μm Millipore filter, sonicated for 50 min, and degassed with the instrument degasser. The setting of the RI detector was optimized, with a temperature setting of 30°C. The signal was recorded as a chromatogram. Sugar concentrations in the urine samples were estimated in 91 patients with active UC (recruited for the clinical trial).

Data were analyzed using the Statistical Package for the Social Sciences software for Windows, version 17.0 (SPSS Inc., Chicago, IL, USA). Data were expressed as the median (range) or mean ± standard deviation. Nonnormal continuous variables were analyzed using a nonparametric test (Wilcoxon rank-sum test). The correlation coefficient was used to assess the correlation of sugar ratios with disease activity. P < 0.05 was considered statistically significant.


 » Results Top


The response of the RI detector was linear, with a clear resolution of all sugars, as shown in [Figure 1]. The sugars were first resolved in a deionized water solvent system [Supplementary Figure 1].
Figure 1: Separation of sugar probes on Waters Sugar-Pack column: (A) Sucrose, (B) Lactulose, (C) Mannitol, and (D) Sucralose

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Linearity/calibration curves

The calibration curves were prepared in the urine matrix with the control sample (urine from a healthy volunteer) and found to be linear in all the cases (sucrose: Y = 4278.6x − 247882, R2 = 0.9926; lactulose: Y = 5597.1x − 60329, R2 = 0.9966; mannitol: Y = 55291.1x + 136615, R2 = 0.9973; and sucralose: Y = 5116.4x + 255353, R2 = 0.997). Each point was determined from the mean of three determinations.

The calibration curves are shown in [Figure 2] (calibration curve of sugars in the urine matrix). The lower LOD was 78.838 mg/L for sucrose, 84.994 mg/L for lactulose, 74.789 mg/L for mannitol, and 50.908 mg/L for sucralose. The respective LOQs for these sugars were 262.79 mg/L, 283.31 mg/L, 249.30 mg/L, and 169.69 mg/L, respectively. Thus, sucralose is most sensitive, followed by mannitol, sucrose, and lactulose.
Figure 2: Calibration curves of sugars in urine matrix

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Interferences

The control urine was run as the sample and there was no interfering peak where the sugars of our interest gave their respective signals.

Accuracy

The analytical recovery of sucrose ranged from 94% to 136%. Lactulose had an analytical recovery of 95%–130%. The respective values for mannitol and sucralose were 99%–146% and 101%–129%, respectively. The values are shown in [Table 1].
Table 1: Analytical recovery of sugars in urine matrix

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Precision

The total imprecision of the method (within day) was assessed by analyzing the sugars at two different concentrations six times per day. The percent CV range was calculated for sucrose (2.09%–22.1%), lactulose (0.45%–9.61%), mannitol (5.60%–6.61%), and sucralose (8.15%–12.35%). The inter-day variation was calculated for 7 days at different weekly intervals. Sucrose has 11.95%, lactulose has 4.76%, mannitol has 8.1%, and sucralose has 6.68% of the CV for intraday run [Supplementary Table 1].



Patient data

The mean age of the 91 patients was 34 years, 50% of whom were male. The mean body mass index was 21.52 ± 3.83 and the mean disease duration was 48 months. The baseline distribution of patients by activity, duration, and behavior of the disease is shown in [Table 2]. Disease activity was moderate to severe with an average Mayo score of 9.6 ± 1.2. The onset of the disease was either insidious or acute. Acute onset of UC occurred in 15 patients, while the disease onset was gradual in 76 patients. The majority of the patients (71) had the relapsing and remitting disease course.
Table 2: Disease characteristics of ulcerative colitis patients

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Changes in permeability levels in the patient samples

The chromatogram for the patient (control) urine sample is shown in [Figure 3]. All four carbohydrates were resolved distinctively with clearly distinguishable values for their retention time without any noise. The area under the curve was used to estimate the concentration [Figure 4]. The ratios of the sugars in the urine are shown in [Table 3]. Gastric permeability, IP, and colonic permeability were used to validate the results in patients with UC. Historical controls were used to validate gut permeability.[19],[20],[21] The findings were validated using percent sucrose excretion, LMR, percent sucralose excretion, and SMR in the UC population. In addition, a one-sample Wilcoxon signed-rank test was performed. A significantly higher percent sucrose excretion, LMR, percent sucralose excretion, and SMR were found in UC patients compared to historically healthy controls. The data are tabulated in [Supplementary Table 2].
Figure 3: Chromatogram of the sugars in control (urine sample): (A) sucrose, (B) lactulose, (C) mannitol, and (D) sucralose

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Figure 4: Chromatogram of the sugars in the patient (urine sample): (A) sucrose, (B) lactulose, (C) mannitol, and (D) sucralose

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Table 3: Percentage excretion of urinary sugars and their ratios

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 » Discussion Top


GI mucosa is the first line of defense against immunogenic and pro-inflammatory reactions. Gut epithelial integrity and selective permeability are essential for optimal health. A number of GI diseases are usually examined by endoscopy for mucosal disintegrity, which causes a lot of discomfort for the patients. Other investigations are nonspecific. Intestinal diseases have been found to have damaged mucosa with abnormal permeability. Hence, there is increased absorption of large molecules across the leaky gut mucosal lining and impaired transcellular transit.[4],[5],[6] Assessing permeability in terms of ratios of different sugar moieties based on their metabolic resistance is an excellent noninvasive approach. It has been reported that these sugar ratios can compensate for differences in intestinal transit, volume of distribution, and correction for incomplete urine collection.[22] Therefore, a noninvasive method to assess the IP of different parts of the gut is a valuable advancement for investigations related to GI diseases.

The current HPLC method is fast, precise, simple, and accurate. The amino cartridges were used to prevent damage and to avoid the column performance degradation. In addition, the method also eliminates the interfering substances in the chromatograms. Sample drying effectively helped eliminate the false peak of methanol used for charging the cartridge. The current HPLC method is a comparatively simple method than cation exchange resins. The resins usually need to be charged and washed before being used for sample preparation.[16] However, sample preparation is effortlessly simple and less time-consuming with a currently used method. The amino cartridges have effectively increased the performance of the analytical column. There was no variation in either the retention time or the shape of the peaks in the chromatograms after 1000 injections.

The calcium-ethylenediaminetetraacetic acid cation exchange gel column was used in the current analysis to separate the sugars and has a distinct advantage over the amino-bonded silica column in that discrete peaks are obtained.[11] Proper cleaning recommended in the manual proved beneficial and resulted in an increased column shelf life. During method development, another divinylcalcium-bonded carbohydrate-specific column was also used for sugar separation. However, the desired resolution of the sugar peaks was not achieved. Thus, the calcium-bonded cation exchange column has the advantage of clear peak resolution, stability, reproducibility, and high performance.

The analytical recovery rate of the sugars of interest was also good, i.e., 94%–146% in the urine matrix. The precision and accuracy of the current method are tabulated in [Table 2]. Using an internal standard could also have increased precision, e.g., arabinose has a different retention time than the sugars of our interest. Therefore, it may have served as an internal standard. However, since effective separations with good reproducibility were already achieved in the analysis, no internal standard was used.

A RI detector has made the analysis very easy and effective for automation. Linear peaks were obtained with no baseline drift and were compatible with the abundantly available deionized water eluent. It was easily adaptable for routine use and had a good sensitivity. However, it required equilibration and was temperature sensitive. Interestingly, the detection limits were analogous to the ELSD and the PAD systems for lactulose and mannitol (10–20 mg/L).[11] Therefore, the data obtained with the current method were similar to the data obtained with PAD, which represents the simplest protocol to detect all the four sugars in a single injection.[16] However, the disadvantage of the RI detector is that it cannot be used in gradient elution protocols. Nevertheless, this simple method of detecting all sugars in a single analysis with water as the solvent has a distinct advantage of clear peak resolution in the chromatogram. No toxic solvents were used as eluents.

In the current study, the main objective was to detect four sugars: sucrose, lactulose, sucralose, and mannitol. Sucrose is degraded by sucrase shortly after it reaches the small intestine and therefore evaluation of urinary sucrose can serve as a useful marker of gastroduodenal permeability. The reliability of the test is based on the rapidity of the hydrolysis of sucrose in the small intestine.[23] In addition, percent sucrose excretion is an established marker for gastroduodenal permeability.[18]

For the assessment of IP, the preferred sugars used in the small probes are mannitol and, in the large probes, sucralose and lactulose.[24] Mannitol, an easily absorbable monosaccharide, serves as a marker for transcellular uptake. After oral administration, extremely small amounts of lactulose and sucralose are passively taken up into the intestinal mucosa via the paracellular pathway and excreted unchanged in the urine.[20],[25] This may serve as an important marker of mucosal integrity.[25] The brush border of the small intestine cannot digest either mannitol or lactulose and therefore these two probes can be used in combination to detect IP.[19] The LMR is a validated measure of small bowel permeability.[18] The permeation of lactulose depends on the permeability through the pores.[2]

However, colon bacteria normally ferment lactulose, and in contrast, both sucralose and mannitol are not metabolized.[19],[26] In addition, sucralose is impervious to the activity of sucrase and is passively absorbed in the small and large intestines. It is then excreted in the urine. Therefore, it has been used to assess colonic permeability.[3] Since sucralose and mannitol remain available for absorption throughout the gut, their ratio can also serve as a measure of the permeability of the whole gut.[19]

In the present study, the increased gastroduodenal permeability compared to historical controls is indicative of leaky gut in patients with active UC. The increased excretion of sugars may be due to this leaky state and severe ulcer conditions reported in IBD and celiac disease.[27] Several studies have also reported that the increased or abnormal IP is mainly associated with the increased activity index of the disease.[27],[28] The results have validated our methodology and its usefulness in the UC population.

The apparent advantage of the current RI-HPLC method is that the technique is simple and reliable. However, there were a few limitations. Historical controls were used to corroborate the results.


 » Conclusion Top


The current study presents a highly efficient analytical method using HPLC equipped with an RI detector to detect four significant carbohydrate moieties in urine samples from UC patients. The technique is rapid, sensitive, easily automated, and reliable for the simultaneous determination of sugars as probes of the impaired IP in UC patients.

Acknowledgments

The Department of Biotechnology, Ministry of Science and Technology, Government of India (DBT Project No. BT/PR14971/20/507/2010), sponsored this study.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Adenis A, Colombel JF, Lecouffe P, Wallaert B, Hecquet B, Marchandise X, et al. Increased pulmonary and intestinal permeability in Crohn's disease. Gut 1992;33:678-82.  Back to cited text no. 28
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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