KR20090045603A - Diagnostic method for galactosemia using high performance anion-exchange chromatography and pulsed amperometric detection - Google Patents

Abstract

The present invention relates to a method for diagnosing galactosemia by quantifying galactose and monophosphate galactose using high performance anion exchange chromatography and pulsed electrochemical detectors. The galactosemia diagnosis method of the present invention has a shorter analysis time than the conventional method, reduces the risk of misdiagnosis through the complete separation analysis of galactose and glucose, monophosphate and monophosphate galactose, and is the most common type of galactosemia. Reliable diagnosis of galactosemia of type 1 and type 2 is possible. In addition, since quantification is possible, it is possible to determine the severity of the disease, to control the degree of treatment, and to determine the prognosis thereof.

Galactosemia, high performance anion exchange chromatography, pulse electrochemical detector, galactose, galactose monophosphate

Description

Diagnostic method for Galactosemia using high performance anion-exchange chromatography and pulsed amperometric detection

The present invention relates to a method for diagnosing galactosemia using high performance anion exchange chromatography and pulsed electrochemical detectors.

Galactoseemia is a rare genetic disease that is caused by a lack of important enzyme metabolism in the human body, resulting in the accumulation of galactose and its metabolites in the body. It is a disease that eventually causes liver cirrhosis after showing delay. Galactoseemia refers to galactose-1-phosphate uridyltransferase (GALT deficiency), type 2 galactokinase deficiency and type 3 galactoseemia (uridine), depending on the enzymes involved in the galactose metabolism at each stage. diphosphate galactose-4-epimerase deficiency). Type 1 galactoseemia accumulates galactose and its metabolite, gal-1-phosphate (Gal-1-P) in the body due to a deficiency of GALT, an enzyme that breaks down galactose into glucose, and type 2 galactosease (galactokinase) binds to the accumulation of galactose in the body. This causes abnormalities in the glandular cells of the kidneys, liver and brain, and all foods containing normal milk (milk powder and breast milk) or galactose are toxic in the body.

For the diagnosis of galactosemia, the Beutler method for measuring enzyme deficiency, the Paigen method for semi-quantifying galactose and metabolites in the body by biological inhibition therapy, and the enzyme colorimetric method Enzymatic colorimetric methods have been used to qualitatively quantify galactose in the blood. However, these assays require special reagents and require constant temperature and humidity regulation under experimental conditions. In addition, because the results are semi-quantitative, other methods of diagnosis are needed to determine the exact disease level. In addition, the enzyme colorimetric method is the most widely used, but when there is a material that shows a similar color in UV absorption, it has a disadvantage that it is difficult to distinguish from the interfering material.

The most important problem in the quantification of galactose in the blood is the separation of glucose and monophosphate that are present in excess in the blood. Glucose and monophosphate glucose are structurally very similar as epimers of galactose and monophosphate galactose and a fourth carbon, and have a slight difference in pKa values, making them difficult to distinguish in general analysis. In the Butler and Paigen methods described above, there is a risk of misdiagnosis due to glucose and monophosphate glucose.

In the analysis using high performance liquid chromatography (HPLC), the analysis of galactose and glucose using an amino column and a refractive index (RI) detector is commonly used, but the sensitivity is low in mmol / L and the trace components in the blood are low. It is impossible to measure

In addition, the analysis on the reversed phase column through fluorescence derivatization has improved sensitivity compared to RI, but since the derivatization step is complicated, the processing time is long, and the step is complicated, there is a large amount of error in the trace analysis because the loss is present. There are disadvantages. In addition, the analysis takes about 20 minutes, which is less efficient than other diagnostic methods.

 Pulsed electrochemical detectors have an electrical property at a high pH by using the difference in the fine pKa value of sugars, thereby enabling analysis of sugars having almost the same structure and properties. In the diagnosis of galactosemia, the present inventors skip the derivatization process and use high-performance anion exchange chromatography and pulsed electrochemical detectors to shorten the analysis time, and analyze the galactose having excellent sensitivity through complete separation of glucose and phosphorylated sugars. The present invention has been completed by the development.

An object of the present invention is to provide a method for diagnosing galactosemia by simultaneously quantifying galactose and monophosphate galactose in blood.

In order to achieve the above object, the present invention provides a combination of high performance anion exchange chromatography and pulsed electrochemical detector which is very effective for quantifying carbohydrates in nature or in blood.

The high performance anion exchange chromatography analysis method uses sodium hydroxide solution as a mobile phase and a mixed solution of sodium hydroxide solution and sodium carbonate solution as a gradient elution method to completely separate the peaks of galactose and glucose while simultaneously working. The peaks of galactose and monophosphate glucose are also completely separated to provide simultaneous quantification of galactose and galactose monophosphate.

The present invention also provides a new method for diagnosing galactosemia using blood buried in a Guthrie Card instead of directly analyzing galactose in the blood.

 The analysis method of galactose using high performance anion exchange chromatography and pulsed electrochemical detector according to the present invention shortens the analysis time and reduces the risk of misdiagnosis through complete separation analysis of galactose and glucose, monophosphate and galactose monophosphate. It is possible to reliably diagnose galactosemia of type 1 and type 2, the most common type of galactosemia.

In addition, galactosemia diagnosis method using the blood buried in the room has the advantage that it is very convenient for the collection, handling, transport and storage of samples.

The galactosemia diagnosis method of the present invention is a method of simultaneously quantifying galactose and monophosphate galactose in blood using high performance anion exchange chromatography and a pulsed electrochemical detector. In the first step, the protein-free sample was used as the sodium hydroxide solution, and the mixed solution of sodium hydroxide and sodium carbonate as the mobile phase. And a third step of detecting galactose and monophosphate galactose. More detailed description of the galactosemia diagnosis method of the present invention is as follows.

1. Step 1: Sample Processing Step

The sample blood is buried in the filter paper, and the dried filter paper is sampled with a piece of about 3.2 mm in diameter (2.00 ± 0.0005 mg, n = 20). Each sample is sealed by adding 95 μL of 1% (w / v) trichloroacetic acid (TCA) as the extraction solvent. Extraction is carried out for 30 minutes in a shaker. After addition of 10 μL of 10% (w / v) TCA for alveolar protein, resealed and centrifuged at 12,000 rpm for 10 minutes, the supernatant is taken and filtered with a MFS 13 syringe filter.

2. Step 2: Analyze Phase

A solution prepared by mixing 10 mM sodium hydroxide solution A for monosaccharide analysis and 10 mM sodium hydroxide and 25 mM sodium carbonate for phosphorous analysis at a mixing ratio of A: B (v / v) was used as a mobile phase. It was analyzed at a flow rate of 1 mL / min in the gradient elution conditions shown in Table 1.

The column used an anion exchange column 4 × 250 mm Carbopac PA1 and 4 × 50 mm Carbopac PA1 Guard. The analytical solvent was subjected to reduced pressure filtration and sonication, and helium gas was injected during the analysis time, thereby preventing carbon dioxide in the air from affecting the reproducibility of the retention time.

TABLE 1

Minutes A: 10 mM NaOH B: 10 mM NaOH + 25 mM Na ₂ CO ₃ 0 100 0 3 100 0 7 20 80 13 20 80 15 100 0 35 100 0

3. The third step: detection step

The pulsed electrochemical detector uses Dionex's 4000i model with Au-Flow cells, stainless steel auxiliary electrodes and Ag / AgCl standard electrodes as active electrodes. Operating potential E1 = + 0.05 V; t1 = 0.4 s, E2 = + 0.75 V; t2 = 0.3 s, E3 = -0.20 V; With t3 = 0.6 s, the sensitivity of the detector was 1 μA full-scale and the analytical results were processed by the Dschrom program provided by Donam Instruments.

The reagents used in the examples of the present invention are as follows. Galactose, glucose, monophosphate galactose, monophosphate glucose, 50% sodium hydroxide (NaOH), sodium carbonate, and trichloroacetic acid (TCA) were purchased from Sigma (St. Louis, MO). Other reagents and reagents used were certified above the analytical grade. For the preparation of standard samples, both the sample solution and the instrument mobile phase used a laboratory distillation unit and an 18 MΩ purification unit Automatic aquarius AW-1001 (Top Trading Co., Seoul, Korea). Solution filtration was performed using a Millipore membrane filter (type HA, pore size 0.45 μm). All samples were filtered prior to instrument injection using a MFS 13 disposable syringe filter.

Preparation of standard blood samples for quality control for an embodiment of the present invention is as follows. For the standard samples used for the establishment of the assay, galactose was purchased from Bio-Rad (UK) containing concentrations of 4.67, 10.59, 20.48 and 53.46 mg / dL. After adding the monobasic galactose standard to the blood of 3, 5, 10, 20, 50 mg / dL, the gusri paper was absorbed, and dried at room temperature in the shade to obtain a standard blood sample.

The actual blood sample for the embodiment of the present invention was provided with 190 normal Korean neonatal samples in a hospital as a control group of galactosemia patients. Thirteen positive samples diagnosed with galactosemia by enzyme colorimetry were provided by the Genetic Metabolic Disease Analysis Team at the Seoul Institute of Science and the King faisal specialist hospital & research center in Saudi Arabia. All blood samples were buried in gooseberry paper and stored at -20 ° C until analysis.

Example

Example 1

A calibration curve for quantifying galactose concentration was presented and the concentration was quantified through this. The method for this was completed through the three steps of sample processing, analysis and detection.

The analytical sample was prepared by diluting 10 mg / mL stock solution with distilled water at a concentration of 1.25, 2.5, 5, 10 and 25 mg / L, respectively. The analysis was performed three times for each concentration to obtain a calibration curve (Table 2). As shown in Table 2, it can be seen that the analysis method of the present invention shows good linearity.

TABLE 2

Of these, 5 mg / L of sample was determined, and the measurement was repeated six times to confirm the reproducibility (Table 3). As shown in Table 3, the mean value of 5 mg / L shows SD (standard deviation) of 0.08 in both galactose and monophosphate galactose, and RSD of 1.52 and 1.62%.

TABLE 3

In addition, inter-day fluctuations were measured over three days for concentrations of 1.25, 2.5, 5 and 10 mg / L (Table 4). Both galactose and monophosphate galactose showed 99.2 ~ 100.4% reproducibility. The relative standard deviation (RSD) is also within 1.17% for galactose and within 1.47% for galactose, indicating excellent reproducibility.

TABLE 4

Example 2

Galactose, glucose, galactosyl phosphate, and glucose monophosphate were mixed and analyzed using the same analytical method as in Example 1 (FIG. 1). As shown in FIG. 1, the complete separation of glucose and monophosphate, which are the major substances causing misdiagnosis in galactosemia, allows accurate diagnosis of galactosemia of type 1 and type 2 as well as accurate quantification of galactose and monophosphate galactose. You can see that it is possible.

Example 3

The analysis conditions were applied to the actual blood sample to measure the recovery. After the blood pretreatment of the above 1-3 steps by adding the standard concentration galactose and monophosphate galactose to the blood of a normal person, it was analyzed. The concentration of the standard used in the experiment was set to 2 mg / L for galactose, 10 mg / L for galactosyl phosphate. The results are shown in Table 5. Galactose averaged 1.95 mg / L with 97.35% recovery and RSD was 2.42%. Monophosphate galactose averaged 9.66 mg / L with 96.56% recovery and 2.39% RSD.

TABLE 5

Example 4

The effectiveness of the assays of the present invention was evaluated for normal samples and galactose and monophosphate galactose positive samples provided from frontline hospitals and neonatal screening laboratories. In the normal blood sample shown in FIG. 2A, peaks of galactose and monophosphate galactose were hardly detected. However, in the galactose and monophosphate galactose positive samples of (B), the levels of galactose and monophosphate galactose were remarkably high compared to the normal, and it can be seen that the accuracy can be estimated by the accurate values.

As described above, the galactosemia diagnosis method of the present invention has excellent reproducibility, resolution, and high recovery rate. In addition, a quick and simple pretreatment is possible by omitting the derivatization step used in the existing galactose analysis method, and it is convenient to store and move the sample because it is a method using a margin. Unlike the conventional method for diagnosing galactosemia, only negative and positive determinations through the semi-quantitative method, the galactosemia diagnosis method of the present invention can determine its degree through accurate values. In congenital metabolic diseases, accurate judgment and quick action are the most important. The method of diagnosing galactosemia according to the present invention can be said to be a good way to determine the prognosis with the control of the degree of treatment through accurate values.

1 is a high performance anion exchange chromatography by gradient elution using a sample containing galactose, glucose, galactose monophosphate and monophosphate glucose as a mobile phase with a 10 mM sodium hydroxide solution and a mixed solution of 10 mM sodium hydroxide and 25 mM sodium carbonate. It is the chromatogram detected using the pulse type electrochemical detector. (1: galactose, 2: glucose, 3: monophosphate galactose, 4: monophosphate)

2 is a high performance anion exchange chromatograph using gradient elution with a normal blood sample (A) and galactose and galactose monophosphate positive sample (B) as a mobile phase with a 10 mM sodium hydroxide solution and a mixed solution of 10 mM sodium hydroxide and 25 mM sodium carbonate. Chromatogram detected using chromatography and pulsed electrochemical detector. (1: galactose, 2: galactose monophosphate)

Claims (8)

A method of diagnosing galactose in blood using high performance anion exchange chromatography and pulsed electrochemical detectors, (a) removing the protein from the blood sample; (b) passing the analytical column by gradient elution using a protein-free sample as a sodium hydroxide solution and a mixed solution of sodium hydroxide and sodium carbonate as a mobile phase; And (c) detecting galactosemia comprising detecting galactose and monophosphate galactose in the blood using a pulsed electrochemical detector.  The method for diagnosing galactosemia according to claim 1, wherein helium gas is injected into the mobile phase to prevent contamination of the carbonate ions in the mobile phase. The galactose according to claim 1 or 2, wherein the mobile phase has a sequential gradient elution of a volume ratio of sodium hydroxide solution: mixed sodium hydroxide and sodium carbonate solution from 100: 0 to 20:80 and again to 100: 0. How to diagnose bloodemia. The method of claim 1 or 2, wherein the flow rate of the mobile phase is 1 mL / min. 4. The method of claim 3, wherein the flow rate of the mobile phase is 1 mL / min. A method for separating galactose and monophosphate galactose by gradient elution from a sample containing galactose and galactose monophosphate using high performance anion exchange chromatography using a sodium hydroxide solution and a mixed solution of sodium hydroxide and sodium carbonate as a mobile phase.  7. The method of claim 6 wherein gallium and monophosphate galactose are separated from the sample, characterized by injecting helium gas to prevent contamination of the carbonate ions in the mobile phase. 7. The galactose of claim 6, wherein the mobile phase has a sequential gradient elution from 100: 0 to 20: 80 and again to 100: 0 in a sodium hydroxide solution: sodium hydroxide and sodium carbonate mixed solution. Method for separating monophosphate galactose.

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