Method of detecting and diagnosing the progression of diabetes

Abstract

The subject of the invention is the method of detecting and diagnosing the progression of diabetes using Raman spectroscopy which involves the examination of the changes in the composition of urinary extracellular vesicles which confirm the existence of the condition and its progression. The invention can be applied in clinical practice, in particular in the early clinical diagnostics of diabetes and in the monitoring of its progression, in particular diabetic nephropathy and advanced renal impairment caused by diabetes.

Claims

1. A method of detecting and diagnosing diabetes comprising the following steps: a) Urinary extracellular vesicles (UEV) are isolated from the urine sample, b) Raman spectra are registered and the analysis of the distribution of the intensity of characteristic bands is performed on the UEV, c) If it is confirmed that the value of RI for the intensity of the Raman spectrum is lower than the value of RI for the Raman spectrum obtained in an identical way for the sample collected from a healthy individual, the patient is diagnosed with diabetes.

2. The method according to claim 1 wherein the urine sample is subject to centrifugation at 2000×g for about 30 minutes in stage a).

3. The method according to claim 1 wherein, in stage a), the urine sample is concentrated using a dialysis membrane with large-diameter pores permeable for the molecules of the average molecular weight below 1000 kDa (MWCO), which is followed by washing.

4. The method according to claim 1 wherein the washing solution contains silver chloride and sodium dichloroisocyanurate in the amount of 0.1 mg of silver chloride and 4.5 mg of sodium dichloroisocyanurate per litre of the urine sample.

5. The method according to claim 1 wherein the characteristic bands in the Raman spectrum are located in the following ranges: from 980 cm.sup.−1 to 1020 cm.sup.−1, from 1110 cm.sup.−1 to 1140 cm.sup.−1, from 1420 to 1470 cm.sup.−1 and from 1565 cm.sup.−1 to 1710 cm.sup.−1.

6. The method according to claim 1 wherein, in stage c), in order to determine the change in the intensity of the Raman spectrum, RI is calculated on the basis of the following formula: $\mathrm{RI}=\frac{I_{\mathrm{Amid}I}}{I_{Phenylalanin}}\times a+\frac{I_{Lipids}}{I_{\mathrm{Phenylalanin}}}.$

7. The method according to claim 6 wherein, in stage c), the values of RI for the group of healthy individuals are above 1000, from 1144 to 1164.

8. The method according to claim 6 wherein, in stage c), the values of RI for patients with diabetes are below 600.

9. The method according to claim 6 wherein, in stage c), the values of RI for the group of patients with controlled diabetes are from 550 to 600.

10. The method according to claim 6 wherein, in stage c), the values of RI for the group of patients with uncontrolled diabetes are from 390 to 410.

11. The method according to claim 6 wherein, in stage c), the values of RI for the group of patients with diabetic nephropathy are below 600.

12. The method according to claim 6 wherein, in stage c), the values of RI are below 300 for the group of patients with advanced renal impairment caused by diabetes with the glomerular filtration rate (GFR) below 46 mL/min/1.73 m.sup.2.

Description

(1) FIG. 1 shows the microphotograph of urinary extracellular vesicles (UEVs) obtained by means of centrifugation from a healthy individual (C) and a person with uncontrolled type 2 diabetes (UD);

(2) FIG. 2 shows the Raman spectra of the pooled samples obtained from patients with controlled type 2 diabetes (CD), patients with uncontrolled diabetes (UD) and 2 samples from the healthy control group (C);

(3) FIG. 3 shows the principle component analysis (PCA) of the Raman spectra obtained from patients with CD (.square-solid.), UD (.box-tangle-solidup.) and from the control group C (•)—the graphs are in non-nominal units and present the share of the component, a variable in this case (band intensity [a.u.]), in the model of a covariance matrix.

(4) FIG. 4 shows the recording of the averaged spectra for 5 groups of patients classified according to the degree of kidney failure.

EXAMPLE 1

Diagnosing the Presence of Diabetes, Controlled Diabetes and Uncontrolled Diabetes

(5) The Description of the Group Under Examination

(6) The group under examination were patients with type 2 diabetes (n=45). The patients were divided into 2 groups according to the diabetes control level based on the level of glycated haemoglobin (HbA1C) following the 2016 guidelines of the Polish Diabetes Association: the group with controlled diabetes (CD) (n=19) and the group with uncontrolled diabetes (UD) (n=26) where HbA1C>7%. The group of patients was compared with the control group (n=6). Table 1 shows the characteristics of the group under examination.

(7) Additionally, the patients' history was collected, which included demographic data, arterial blood pressure, BMI, dietary habits and addictions (smoking and alcohol consumption) as well as the anti-diabetic treatment and drug therapy (potassium-sparing diuretics, loop diuretics, thiazide and thiazide-like diuretics, beta-blockers, ACEI, Ca antagonists, Na/K ATPase inhibitors, vasodilating diuretics, clopidogrel, acetosalicylic acid and statins), information on the surgeries and surgical procedures undergone by patients, including coronary artery bypass grafts (CABG) and percutaneous coronary interventions (PCI), as well as comorbidities and cancer. The patients diagnosed with the following conditions during the examination or history collection were excluded from the study: diagnosis or suspicion of a recent bacterial or viral infection, cancer, a cardio-vascular incident or surgery within 6 months prior to the history collection, signs of liver impairment, steroid and non-steroid anti-inflammatory therapy, surgical treatment of obesity, hormonal replacement therapy in the case of women, autoimmune diseases (i.e. chronic arthritis, antiphospholipid syndrome).
The Condensation and Isolation of Urinary Extracellular Vesicles from Urine Samples

(8) Samples of morning urine of about 250 ml were collected from the patients and the healthy volunteers. Initially, the urine was centrifuged at 2000×g for about 30 minutes in order to remove epithelial cells, bacteria and urinary deposits. Then, the urine was concentrated using hydrostatic filtration/dialysis (HDF), a method in which urine is filtrated using a dialysis membrane with large-diameter pores permeable to molecules whose molecular weight is below 1000 kDa (MWCO). After volume reduction, the sample was washed using deionised water and, again, reduced to the volume of a few millilitres. In another variant, it is possible to use colloidal silver containing silver chloride and sodium dichloroisocyanurate in the amounts of, respectively, 0.1 mg and 4.5 mg per every litre of the urine sample, in order to chemically inactivate the remaining bacteria.

(9) The Evaluation of the Presence of UEVs Using the Transmission Electron Microscope (TEM)

(10) The deposit samples were centrifuged in Eppendorf tubes and stabilised in 2.5% glutaraldehyde (cat. no. G5882, Sigma-Aldrich, St. Louis, USA) in 0.1 M of cacodylic buffer (cat. no. C4945, Aldrich, St. Louis, USA) for 2 hours in room temperature, and then in 1% solution of osmium tetrachloride (OsO.sub.4) for 1 hour. The samples were dehydrated in ethanol and embedded in PolyBed 812 in 68° C. Snippets for analysis were placed on a mesh (300 mesh grids). Next, the snippets were contrasted using uranyl acetate and lead citrate. The JEOL JEM 2100HT electronic microscope (JEOL Ltd., Tokio, Japonia) with accelerating voltage of 80 kV was used for observation. This stage is presented in FIG. 1.

(11) Raman Spectroscopy and the Analysis of Characteristic Bands

(12) Raman spectra were registered using the Renishaw InVia Raman spectrometer equipped with an optical confocal microscope with the Leica N PLAN EPI dry lens (100×, NA 0.85). The laser emitting the light of the wavelength of 532 nm was cooled using air; the laser power in the sample position was about 15 mW. The CCD detector was cooled thermoelectrically to the temperature of −70° C. A drop of the UEV suspension was placed on the CaF.sub.2 window and left there until the water evaporated. Each dried sample was measured in at least 15 randomly selected places. Eventually, 100 scans with the exposure time of 20 s and the resolution of about 1.5 cm-1 were collected from each place. The spectrometer was calibrated on the basis of the location of the Raman band of the silicon plate inside the device. A principle component analysis (PCA) was carried out using the Unscrambler X 10.3 software (CAMO AS, Norway). Prior to PCA, Raman spectra were adjusted by cutting off the baseline, and, subsequently, they were smoothed and normalised. The registered Raman spectra were presented in FIG. 2. Table 2 shows the intensity of the characteristic bands that are important for the differentiation of individual sample groups, which was concluded on the basis of PCA. FIG. 3 shows the principle component analysis of the Raman spectra registered for the samples obtained from the patients. It clearly shows that individual points gather in clusters that include the individual groups under examination.

(13) TABLE-US-00001 TABLE 1 The characteristics of the groups under examination as regards biochemical and epidemiological parameters Controlled Uncontrolled Control diabetes diabetes (C) (CD) (UD) Parameter n = 6 n = 19 n = 26 p-value Gender (M/F) 6 13/6 18/8 — Age [years] 51 (7) 62 (16) 61 (13) 0.099 HbA1C [%] — 44 (42-48) 68 (62-73) <0.0001 Glucose in serum 5 (5-6) 7 (6-8) 9 (7-11) <0.0001 (mmol/L) Albumin in 6 (4-13) 6 (1-10) 30 (12-244) 0.0002 urine [mg/L] Creatinine in urine 12 (9-16) 5 (4-10) 6 (5-9) 0.008 [mM] Creatinine in 69 (60-85) 73 (62-87) 77 (61-104) 0.546 serum [μM] GFR 84 (76-100) 89 (68-103) 86 (64-100) 0.921 [mL/min/1.73 m.sup.2]

(14) TABLE-US-00002 TABLE 2 A list of characteristic bands for which statistically significant differences were observed in Raman spectra Average band intensity [a.u.].sup.# Band position C CD UD P 1565-1710 cm.sup.−1 0.303 (0.016) 0.223 (0.055)* 0.247 (0.024)* 4.38E−7 Amid I 1420-1470 cm.sup.−1 0.115 (0.006) 0.111 (0.007)  0.138 (0.019)* 3.82E−5 Lipids 1110-1140 cm.sup.−1 0.014 (0.001) 0.015 (0.003)  0.017 (0.003)* 0.024 Proteins 980-1020 cm.sup.−1 0.023 (0.005) 0.034 (0.006)* 0.053 (0.007)* 2.97E−9 Phenylalanine .sup.#Average values of the band intensity have been normalised and are expressed in arbitrary units [a.u.] *intensity values that differ from control values in a statistically significant way for the level of significance α = 0.05; the groups were compared between each other using the non-parametric Kruskal-Wallis test; additionally, the U Mann-Whitney test was applied for both groups.
The Determination of RI—Ratio Intensity—which Differentiates the Group of Patients with Controlled Diabetes (CD) and Uncontrolled Diabetes (UD) from the Control Group (C)

(15) To determine the value of RI, which differentiates the CD and UD group from the control group C in an arbitrary way, the following formula was used:

(16) $\begin{array}{cc}\mathrm{RI}=\frac{I_{\mathrm{Amid}I}}{I_{Phenylala\mathrm{nine}}}\times a+\frac{I_{Lipids}}{I_{\mathrm{Phenylalanine}}}& \left(1\right)\end{array}$
where: I.sub.AmidI—is the value of the band intensity for Amid I (1565-1710 cm.sup.−1) I.sub.Phenylalamine—is the value of the band intensity for phenylalanine (980-1020 cm.sup.−1) I.sub.Lipids—is the value of the band intensity for lipids (1420-1470 cm.sup.−1) a—is the weight factor determined as the quotient of statistical significance (p) for the difference between the intensity of bands I.sub.Amid I and I.sub.Lipids between the group of patients and the group of healthy individuals; in this case a=87.2

(17) $a=\frac{p_{\mathrm{Lipids}}}{p_{\mathrm{Amid}I}}a=87.2$

(18) The probability value (p) was calculated in the OriginPro 2017 programme (according to the algorithm for the Kruskal-Wallis test).

(19) The Kruskal-Wallis test statistics in the OriginPro 2017 programme is calculated according to the following formula:

(20) $H=\frac{12}{N\left(N+1\right)}\underset{j=1}{\overset{k}{.Math.}}\frac{R_j^2}{n_j}-3\left(N+1\right)$ H— the Kruskal-Wallis test N— the total number of observations k—the number of the groups compared n.sub.j—the number of observations in a given group R.sub.j—the total of ranks in a given group
On the basis of the equation, IR for group C, CD and UD was determined:
RI.sub.C=1154±10.5
RI.sub.CD=575±24.7
RI.sub.UD=409±9.9

(21) The values were provided with the approximation error experimentally determined.

(22) On the basis of this, the threshold value for diabetes was determined:
RI<600

EXAMPLE 2

Diagnosing the Progression of Diabetes—Identifying the Presence of Diabetic Nephropathy and Determining its Progression

(23) An additional analysis of the variability of the urinary extracellular vesicle spectra using Raman spectroscopy was performed for the extended group of patients with type 2 diabetes (n=18) and various degrees of renal impairment in diabetic nephropathy defined according to the value of the glomerular filtration rate (GFR).

(24) Group 5<15 ml/min/1.73 m.sup.2 (n=4)

(25) Group 4 15-30 ml/min/1.73 m.sup.2 (n=7)

(26) Group 3 31-45 ml/min/1.73 m.sup.2 (n=2)

(27) Group 2 46-60 ml/min/1.73 m.sup.2 (n=1)

(28) Group 1 61-90 ml/min/1.73 m.sup.2 (n=4)

(29) and the control group>90 ml/min/1.73 m.sup.2 (n=6)

(30) Clinical and epidemiological data can be found in Table 3.

(31) TABLE-US-00003 TABLE 3 The characteristics of the groups of patients under examination as regards selected biochemical and epidemiological parameters. Parameter Control 5 4 3 2 1 p Gender (F/M) 1/5 2/2 2/5 2/0 0/1 2/2 — Age (years) 55 (5) 70 (9) 67 (11) 81 (3)† 56 66 (10) 0.02463 Creatinine in 94 (80-95) 431†* (370-606) 220†* (197-254) 97 (35-45) 126 76* (56-101) 0.00324 serum (μmol/l) GFR 90 (84-111) 11†* (7-12) 23†* (21-26) 40 (35-45) 55 83* (80-87) 0.00237 (ml/min/1.73 m.sup.2) Glucose 5.3 (5.1-5.4) 15.9†* (10.6-19.0) 7.6† (6.8-9.3) 5.7 (5.4-6.0) 6.8 8.2† (7.9-11.1) 0.00758 (mmol/l) hsCRP 0.34 3.3† (2.5-7.1) 6.8† (4.5-10.3) 2.6 (2.5-2.7) 0.9 18.5†* (14.0-23.0) 0.09321 (mg/l) LDL CHOL 3.2 1.9 (1.5-2.7) 3.4 (2.7-3.7) 2.6 (2.5-2.7) 3.4 2.3 (0.9-3.0) 0.10374 (mmol/l) HDL CHOL 1.31 0.7 (0.6-0.8) 0.9 (0.9-1.0) 1.2 (1.1-1.3) 1.5 1.1 (0.8-2.0) 0.08204 (mmol/l) TG 0.7 2.3 (1.3-3.5) 1.4 (1.1-2.8) 1.6 (1.6-1.7) 2.5 1.1 (0.9-1.9) 0.38717 (mmol/l) CHOL 4.8 3.7 (3.74.1) 4.8 (4.3-4.9) 4.5 (4.3-4.9) 6.0 3.6 (2.9-5.5) 0.29512 (mmol/l) Albumin in urine 4.4 (3.9-4.4) 896.9†* 254†* (39.5-2880) 8.8 (2.0-15.5) — 49.6 (30.4-257) 0.0665 (mg/l) The groups were compared between one another using ANOVA or Kruskal-Wallis tests. †a statistically significant difference for the group as compared with the control group *a statistically significant difference for the group as compared with the remaining groups of patients

(32) The table above shows that the patients with the highest level of renal impairment (Group 5 and Group 4) were significantly different from the remaining groups of patients with regard to: blood glucose concentration the concentration of albumin in urine

(33) Next, the Raman spectra were registered (just like before) for the range of 400-1800 cm.sup.−1, for the urinary extracellular vesicles isolated from the morning urine samples (80-100 mL) collected from the patients, individually for each patient.

(34) Then the spectra were divided into 5 groups according to the criterion of renal impairment (GFR), the spectra were averaged and the average spectra for each group were plotted. The graphs can be found in FIG. 4, which shows the recording of the averaged spectra for 5 groups of patients classified according to the degree of kidney failure: 1) 61-90 ml/min/1.73 m.sup.2; 2) 46-60 ml/min/1.73 m.sup.2; 3 31-45 ml/min/1.73 m.sup.2; 4) 15-30 ml/min/1.73 m.sup.2; 5<15 ml/min/1.73 m.sup.2.

(35) On the basis of the spectra determined, the analysis of the average values of the area under the curve (AUC) was carried out for the selected bands representing metabolites present in urinary extracellular vesicles of the patients with diabetes and various degrees of renal impairment. Additionally, the analysis involved the values of the average intensity of the selected bands.

(36) On the basis of the biochemical parameters determined and the analysis of Raman spectra, the correlation analysis using a parametric test (a Pearson correlation test) for the AUC values in individual ranges and the biochemical marker concentration values was performed. On the basis of the calculations performed, it can be concluded that, for the patients with diabetes, the values for the Raman spectrum band corresponding to nucleic acids (DNA) correlate significantly with: eGFR (negative correlation), the concentration of creatinine in serum (positive correlation),
which corresponds to the level of progression of kidney failure.
Moreover, for the patients with diabetes, the values of the Raman spectrum corresponding to: lipids (1282-1305 cm.sup.−1) and proteins and lipids (1403-1502 cm.sup.−1)
correlate significantly (positive correlation) with the concentration of triglycerides (TG), which corresponds to the level of progression of dyslipidaemia in diabetes.
Determining RI to Assess the Degree of Renal Impairment for Averaged Spectra

(37) The additional analyses carried out for the group of patients with diabetic nephropathy which show the application of the introduced RI value to the assessment of the degree of renal impairment in patients with diabetes.

(38) The following equation was used to determine the value of RI, which differentiates, in an arbitrary way, the CD (controlled diabetes) and UD (uncontrolled diabetes—uncontrolled blood sugar level) groups from the control group C:

(39) $\begin{array}{cc}\mathrm{RI}=\frac{I_{\mathrm{Amid}I}}{I_{Phenylalanin}}\times a+\frac{I_{Lipids}}{I_{\mathrm{Phenylalanin}}}& \left(1\right)\end{array}$
where: I.sub.Amid I—is the value of the band intensity for Amid I (1565-1710 cm−1) I.sub.Fenyloalanina—is the value of the band intensity for phenylalanine (980-1020 cm.sup.−1) I.sub.Lipidy—is the value of the band intensity for lipids (1420-1470 cm.sup.−1) a—is the weight factor determined as the quotient of the statistical significance (p) for the difference between the intensity of the bands I.sub.Amid I and I.sub.Lipids between the groups of patients and the group of healthy individuals; in this case a=87.2

(40) $a=\frac{p_{\mathrm{Lipids}}}{p_{\mathrm{Amid}I}}$
a=87.2

(41) On the basis of the area under the curve (AUC), the parameters determined and the average band intensity (I), RI for individual groups of patients (Table 4) was determined using formula (1)

(42) TABLE-US-00004 TABLE 4 Control 5 4 3 2 1 RI calculated for AUC 1722 569 568 524 986 733 RI calculated for I average 331 109 108 100 187 140

(43) On the basis of the RI values calculated, it was established that the threshold value for diabetic nephropathy was RI<600.

(44) The patient with RI<300 is diagnosed with advanced renal impairment with the glomerular filtration rate (GFR) below 46 mL/min/1.73 m.sup.2. 1 van der Pol E, Böing A N, Harrison P, Sturk A, Nieuwland R. Classification, functions, and clinical relevance of extracellular vesicles. Pharmacol Rev. 2012; 64(3); 676-705.2 van der Pol E, Böing A N, Gool E L, Nieuwland R. Recent developments in the nomenclature, presence, isolation, detection and clinical impact of extracellular vesicles. i inni. 1, 2016, J Thromb Haemost. 2016; 14(1); 48-56.

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