EP2577270A1

EP2577270A1 - Method and device for measuring and monitoring concentration of substances in a biological fluid

Info

Publication number: EP2577270A1
Application number: EP20110741089
Authority: EP
Inventors: Ivo Fridolin; Fredrik Uhlin; Jana Jerotskaja; Kai Lauri; Merike Luman
Original assignee: Tallinn University of Technology
Current assignee: Tallinn University of Technology
Priority date: 2010-05-27
Filing date: 2011-05-27
Publication date: 2013-04-10
Also published as: WO2011147425A1

Abstract

Method and device for monitoring removal of hardly or easily diffusible uremic retention solutes during dialysis and for measuring concentration of said substances in a biological fluid, and more specifically in the spent dialysate, for estimation of dialysis dose on dialysis patients. Measurements are performed optically utilizing spectrum of the biological fluid and a concentration calculation algorithm containing the transforming function, including a unique set of optical spectral components at certain wavelengths, to determine the concentration of the substances in specimens in-vitro or flowing fluids on-line. Method and device determines the concentration of the substances in-vitro or on-line utilizing a measuring cuvette suitable for specified measurements. This enables monitoring quantitatively concentrations of substances in a biological fluid and calculation of the dialysis dose for hardly or easily diffusible uremic retention solutes, suitable for automatic and time-efficient way to review the dialysis quality delivered to the end stage renal disease patients.

Description

Method and device for measuring and monitoring concentration of substances in a biological fluid

Technical field

This invention relates to a novel method and a device for measuring and monitoring quantitatively concentrations of substances in a biological fluid and removal of uremic retention solutes utilizing UV-absorbance measurements during dialysis. More specifically, the present invention relates to an optical method utilizing optical spectrum, preferable UV- spectrum and UV-absorbance, preferable UV-absorbance of the spent dialysate, and a specific model, including a unique set of optical spectral components at certain wavelengths, to determine and to monitor, preferable on-line, the concentration and removal of the hardly diffusible uremic retention solutes like protein bound uremic toxins and middle molecules such as beta2-microglobulin (B2M) or easily diffusible uremic retention solutes like urea, creatinine or urea acid having small molecular weight.

Background of the invention

Monitoring of the important biological constituents in biological fluids from the medical point of view can prevent serious pathological conditions and decrease mortality of patients. There is a need for a simple, compact, inexpensive, mobile, reliable method for measuring concentration of water soluble middle and small molecular weight uremic retention solutes like beta2-microglobulin (B2M), urea or creatinine in the spent dialysate for estimation of dialysis dose and nutritional status (protein nitrogen appearance and lean body mass) on dialysis patients.

The uremic syndrome is attributed to the progressive retention of a large number of compounds, which under normal conditions are excreted by healthy kidneys. These compounds are called uremic retention solutes, or uremic toxins, when they interact negatively with biologic functions. The uremic syndrome is a complex„intoxication" of the retention of waste products resulting in multifactorial problems where disturbances in several metabolic functions are reflected in clinical problems. Several organs and organ systems are affected: cardio-vascular system (hypertension, pericarditis and heart failure), peripheral nervous system (polyneuropathy), central nervous system (poor memory, loss of concentration and slower mental ability), hematology (anemia, bleeding tendencies), coagulation, immune status (immunosupression), nausea, vomiting etc.

European Society of Artificial Organs (ESAO) and European Uremic Toxin Work Group (EUTox) have done a lot of research and have had a great success to identify uremic toxins and to connect uremic toxins with the clinical status of the renal patients (Vanholder, De Smet et al. 2003) [22].

In the medical literature the uremic toxins are divided into three groups: 1) small molecules (MW < 500 Da); 2) middle molecules (MW > 500 Da); 3) protein-bound solutes.

Different uremic toxins have effect to the patient by many different ways and extent, and to ensure the best survival, quality of the treatment and the quality of life for the dialysis patients monitoring of several uremic toxins is essential.

Clinically, the most discussed molecules connected to uremic toxicity are the following: Small molecular weight solutes (MW < 500 g/mol): urea, creatinine, uric acid, guanidine - ADMA (asymmetric dimethylarginine), phosphate.

Urea is a small, water soluble molecule with a molecular weight of 60 Da. Urea is the major end product of protein nitrogen metabolism in humans. It constitutes the largest fraction of the non - protein nitrogen component of the blood. Urea is produced in the liver and excreted through the kidneys in the urine. Consequently, the circulating levels of urea depend upon protein intake, protein catabolism and kidney function. Elevated urea levels can occur with dietary changes, diseases which impair kidney function, liver disease, congestive heart failure, diabetes and infection.

The most common method for urea determination is the enzymatic method by the enzyme urease.

The enzyme methodology employed by a specific reagent is based on the following reaction: urea is hydrolyzed in the presence of water and urease to produce ammoniaand carbon dioxide. In the presence of glutamate dehydrogenase (GLDH) and reduced nicotinamide adenine dinucleotide (NADH), the ammonia combines with a-ketoglutarate (a-KG) to produce L-glutamate. The reaction is monitored by measuring the rate of decrease in absorbance at 340 nm as NADH is converted to NAD. To shorten and simplify the assay, the calculations are based on the discovery of Tiffany, et al. that urea concentration is proportional to absorbance change over a fixed time interval.

Creatinine is a de-composition by product of the energy producing compound, creatine phosphate. The amount of creatinine produced is fairly constant and is primarily a function of muscle mass. Creatinine is removed from the plasma by glomerular filtration and then excreted in the urine without any appreciable re-absorption by the tubules. Typically 7-10% of creatinine in the urine is derived from tubular secretion but this is increased in the presence of renal insufficiency. Because creatinine is endogenous and is freely filtered at the glomerulus, it is widely used to assess kidney function (Glomerular Filtration Rate or GFR) and is expressed either as a plasma concentration or renal clearance. Elevated levels of plasma creatinine are associated with impaired renal function. The increase in serum creatinine is the result of uremic retention, but also can be due to muscle breakdown. Morbidity and mortality in hemodialyzed patients are positively correlated with serum creatinine. Creatinine is also a precursor of toxic compound methylguanidine. [1]

Creatinine can be determined in several different ways: 1) Jaffe method [2]; 2) enzymatic colourimetric method.

Jaffe method: testing utilizes picric acid for the Jaffe Reaction to test for creatinine. It forms a colored bright orange-red complex that can be measured using spectroscopy.

The enzymatic colourimetric determination of creatinine which largely eliminates interferences known to the Jaffe method. The series of enzyme catalyzed reactions involved in the assay system are as follows: firstly, the potential interference from endogenous creatine and sarcosine are eliminated by the reaction of creatine amidinohydrolase, sarcosine oxidase and catalase before creatinine is determined. In the final reaction sequence, the formation of the quinoneimine dye product results in an increase in absorbance at 550nm (530-570nm) which is directly proportional to the creatinine concentration in the sample. Ascorbate oxidase eliminates the influence of ascorbate in the sample.

The demerits of the abovementioned methods to determine urea and creatinine include: 1) some compounds similar to urea and creatinine contained in the sample of biological fluid may affect the test accuracy; 2) the operation is complex, needs lots of agents which are hard to keep, and should be operated by professionals; 3) the sample must be de-protein pretreated; and 4) the necessary equipment is expensive.

Uric acid, a final product of the metabolism of purine, is very important biological molecule present in body fluids. It is mostly excreted from human body through the kidneys in the form of urine. The concentration of uric acid in blood increases when the source of uric acid increases or the kidney malfunctions. Hyperuricemia is a symptom when the uric acid concentration is above 7 mg/dL. Uric acid is hard to dissolve in blood and will crystallize when supersaturated. The uric acid crystallites deposit on the surface of skin, in joints, and especially in toes and results in gout. The analysis of the uric acid concentration in blood helps to diagnose gout. In addition to gout, hyperuricemia is connected with lymph disturbance, chronic hemolytic anemia, an increase of nucleic acid metabolism and kidney malfunction. Elevated serum UA contributes to endothelial dysfunction and increased oxidative stress within the glomerulus and the tubulo-interstitium, with associated increased remodeling fibrosis of the kidney [3]. A high level of serum UA, hyperuricemia, has been suggested to be an independent risk factor for cardiovascular and renal disease especially in patients with heart failure, hypertension and/or diabetes [4-6] and has been shown to cause renal disease in a rat model [7]. UA is mostly associated with gout but studies have implicated that UA affects biological systems [8] and also could influence risks of higher mortality in dialysis patients [9] but the pathogenic role of hyperuricemia in dialysis patients is not complete established [10]. High caloric foods and alcohol as well as disturbances of organs and tissues are the main causes of hyperuricemia and even gout. Harm can be prevented and reduced by an early diagnosis and monitoring.

A simple and inexpensive detecting system helps patients to detect the uric acid concentration on their own. As an example, a method and apparatus for the quantitative determination of optically active substances, particularly uric acid, by UV-absorbance, is proposed in WO2009071102 (US60/992156, 04.12.2007), Ivo Fridolin, et al.

Uric acid can be determined in several different ways. Two of the most common methods are (1) reduction method by the reaction of uric acid with alkaline phosphotungstate, and (2) the enzymatic method by the enzyme urease.

In the first method, uric acid is estimated by reducing alkaline phosphotungstate to tungsten blue and measuring the colored product in a colorimeter. The demerits of this method include: 1) some compounds similar to uric acid and ascorbic acid contained in the sample of biological fluid affect the test accuracy; 2) the operation is complex, needs lots of agents which are hard to keep, and should be operated by professionals; 3) the sample must be de- protein pretreated; and 4) the necessary equipment is expensive.

The second method detects uric acid by optical colorimetry and electrochemistry and is classified into uricase-ultraviolet absorption, uricase-peroxidase, uricase-catalase and uricase-electrode methods, wherein the former three methods make use of the color of reaction products and quantitatively detect uric acid of by colorimetry. The decrease in absorbance is proportional to the amount of uric acid initially present. The automatic bio- analyzers used in central bio-laboratories of hospitals detect uric acid by optical colorimetry. An improving system for quantification of biochemical components in biological fluids during analysis where a component reacts with aft analyte is described in US6121050, 19.09.2000 HAN, CHI-NENG ARTHUR.

The blood sample should be pretreated to be serum or plasma first. The merits of the automatic bio-analyzers reside in mass detecting, automation and quickness. However, an automatic bio-analyzer cannot be applied in household detecting because it requires professionals to operate, is expensive, and is particularly hard to store the detecting agents. The uricase-electrode method detects uric acid by electrochemistry. The electrodes can be divided as enzymatic and non-enzymatic. The former produced by a complex production process is hard to store and thus is only suitable for research.

The blood sample should be pretreated to be serum or plasma first. The merits of the automatic bio-analyzers reside in mass detecting, automation and quickness. However, an automatic bio-analyzer cannot be applied in real-time and household detecting because it requires professionals to operate, is expensive, and is particularly hard to store the detecting agents.

Middle molecules (MM) (MW > 500 g/mol): P2-microglobulin, cytokines (interleukin 6), parathyroid hormon (PTH) - (at the same time belongs to the protein-bound group).

Protein-bound solutes: indoxyl sulfate, homocysteine, p-cresol, AGE products, hippuric acid.

Nowdays, due to availability of highly convective dialysis therapies like HemoDiaFiltration (HDF) which target to remove more efficiently the middle molecules (MM) (MW > 500 g/mol), the quality should be assessed by a marker molecule which belongs into MM uremic toxin group (e.g. p2-microglobulin), or behaves like MM as can be expected by several protein bounded uremic toxins. Lack of renal elimination of B2M in patients with end-stage kidney disease results in high level of serum concentrations and could attribute the cause of dialysis -related amyloidosis (Driieke TB, Massy ZA et al 2009) [15] and predict mortality in dialysis patients (Cheung AK, Rocco MV et al 2006 [12]; Cheung AK, Greene T et al 2008 [13]). Reduction of the accumulation and lower long-term levels of these compounds may prevent or delay the appearance of such complications and mortality. β2 -microglobulin (B2M) (MW 1 1 818 D) is the light chain of HLA class I complex and as such is expressed on all nucleated cells. B2M is normally found in low concentrations in the plasma. In end stage renal failure its concentration increases markedly secondary to reduced renal elimination. Uremia-related amyloid is to a large extent composed of B2M and is essentially found in the osteoarticular system and in the carpal tunnel. Uremia related amyloidosis becomes clinically apparent after several years of chronic renal failure and/or in the aged. B2M has become a frequently used marker for the dialytic removal of middle molecules. Behavior of B2M during dialysis is, however, not necessarily representative of that of other middle molecules. Hemodialysis with large pore membranes results in a progressive decrease of predialysis B2M concentrations and in a lower prevalence of dialysis-related amyloidosis and/or carpal tunnel syndrome. In a subanalysis of the Hemodialysis (HEMO) Study, serum B2M levels were directly related to patient outcome. European Best Practice Guidelines (EBPG) have pointed out that despite no surrogate molecule has been identified yet with the characteristics of an ideal marker for MM uremic toxins, B2M is representative in its kinetic behaviour of other MM and peptides of similar size, and may be used as a marker for such molecules. (ERA-EDTA 2002) [16].

B2M is mainly determined by ELISA assay method. Although the method is automated as an automatic bio-analyzer, the merits of the ELISA itself reside in mass detecting discrepancy and complicacy of the method. It cannot be applied in routine or household detecting because it requires professionals to operate, is expensive, and there is hard to store the detecting agents. Great care has been taken to ensure the quality and reliability of the method but however, it is possible that in certain cases unusual results may be obtained due to high levels of interfering factors.

Many MM and protein bound uremic toxins are determined utilizing the high performance reverse liquid chromatography (HPLC) method. For example, indoxyl sulfate has been determined by fluorescence detection (excitation 280 nm, emission 340 nm), and hippuric acid has been analyzed by ultraviolet detection at 254 nm in the serum and in the spent dialysate (Dhondt, Vanholder et al. 2003) [14]. The demerits of this method include: 1) separation of the compounds may be difficult due to similar properties which affects the test accuracy; 2) the operation is complex, needs lots of agents and should be operated by professionals; 3) the sample needs pretreatment for deproteinization; and 4) the necessary equipment is expensive.

Another method for measuring concentration of a substance in a solution having an absorption at 300 nm or less is described in US20080304048A1. However, the method utilizes separation by chromatography which is not suitable for on-line monitoring of the uremic retention solutes.

Another method utilizing UV spectrometer aiming to measure the biocide, sanitizing solutions and quaternary ammonium cation is described in US7372039B2. However, the method utilizes specific wavelengths suitable only for abovementioned chemicals and not for uremic retention solutes in the biological fluids.

Another methods utilizing absorption spectra and spectrophotometric analysis in a liquid sample are described in US201 10040494 A 1 and EP1070954B1. However, the methods utilize library of the reference data as basis for the analysis which is not applicable for the for measuring and monitoring concentration of uremic retention solutes in a biological fluid. Additionally, photo-oxidation of the sample is not valid for uremic retention solutes being an important step in EP1070954B1.

Another method for determination of the amount of waste products in the dialysis liquid during dialysis treatment to control the dialysis machine in order to adapt the dialysis treatment to the patient is described in US6666840, 23.12.2003, Falkvall et al, and in the reference (Fridolin, Magnusson et al. 2002) [17]. The measurements of a concentration of a certain substance or a combination of substances in the dialysis liquid are obtained continuously or regularly on a sample from outgoing dialysis liquid from a dialyzer during dialysis treatment. The measurements are performed spectrophotometrically by means of UV-radiation (wavelength in the range 180-380 nm). At least one parameter for the dialysis treatment is adjusted depending on the measurement of the concentration of the substance or combination thereof. The merits of the described method are that it does not need blood samples, no disposables or chemicals, and is fast. However, the described method is general and does not specify methodology to measure exclusively a single compound and is meant to apply only for dialysis monitoring. Moreover, no results about the concentration measurements are presented. More exact description about the uric acid and urea measurements using the abovementioned method is given in a scientific papers (Uhlin, Lindberg et al. 2005) [21], (Uhlin, Fridolin et al. 2005) [20]. Methods for determination of the dialysis dose as the Kt/V value during hemodialysis treatment using the slope of the natural logarithimic UV absorbance values vs time or the raw UV absorbance values are described earlier (Fridolin, Magnusson et al. 2002 [17]; Uhlin, Fridolin et al. 2003) [19], (Luman, Jerotskaja et al. 2009) [18]. Related method describing estimation of abovementioned parameters by recirculating the dialysis liquid against the blood flowing through the kidney substitution until an mass transfer equilibrium is obtained for both fluids enabling evaluate the initial blood waste concentration is described in EP2005982A1 , 20.06.2007, Castellarnau, A.

Another method relates to a method for dialysis monitoring method and apparatus using near infrared radiation, described in W09819592, 14.05.1998, RIO GRANDE MEDICAL TECH INC. The merits of the described method are similar to that of the UV-radiation. However, the described method measures urea and creatinine by utilizing near infrared radiation spectrometry with different technical and optical considerations. For near infrared radiation spectrometry the principial component analysis using calibration and prediction stage is described in US5886347. Another method, described in RU2212029, 10.09.2003, VASILEVSKIJ A M et al, relies on the Beer-Lambert law and utilizes the millimolar extinction coefficients of the components in the spent dialysate. The example given in this invention describes concentration determination of urea, phosphate, creatinine and uric acid. However, the example is given only for one dialysis session which is a serious limitation and can not be applied for the general use. Furthermore, urea and phosphate do not absorb UV-radiation as incorrectly claimed in this application, and thus concentration measurement of urea and phosphate is impossible using this invention. Moreover, because of several unknown chromophores in the spent dialysate the concentration measurement of creatinine is not applicable using the Beer- Lambert law.

Thus, there is a need for a new method which can directly and easily monitor removal of uremic retention solutes during hemodialysis andperform quantitative concentration measurements of water soluble middle and small molecular weight uremic retention solutes in the biological fluids (e.g. in the spent dialysate) for estimation of nutritional status (protein nitrogen appearance and lean body mass) on dialysis patients and which avoids the disadvantages caused by the analysis in a laboratory.

Moreover, a parameter Lean Body Mass (LBM), suitable for automatic and time-efficient way to review the muscle mass and protein nutritional status of the HD patients, can be monitored by abovementioned novel method for quantitative concentration measurements of uremic retention solutes in the biological fluids.

Additionally, a new, simplified mapping of dialysis dose and nutrition is proposed by using only two parameters - Kt/V and nPNA, suitable for automatic and time-efficient way to review the performed HD strategy based on the data from on-line dialysate side dialysis monitors. The abovementioned novel method for quantitative concentration measurements of uremic retention solutes in the biological fluids combined with the new, simplified mapping of dialysis dose and nutrition, gives an added value for the real-time, automatic monitoring of dialysis patients.

Summary of the inventions

The purpose of the invention is, therefore, a new method and a device for measuring concentration of substances (e.g. middle molecular weight solutes - (MM) (MW > 500 g/mol): p2-microglobulin, Cytokines (Interleukin 6), Parathyroid hormon (PTH) - at the same time belongs to the protein-bound group, small molecular weight solutes - (MW < 500 g/mol): Urea, Creatinine, Uric acid, Guanidine -ADMA (asymmetric dimethylarginine), Phosphate) in biological fluids and monitoring removal of the said hardly and easily diffusible uremic retention solutes during dialysis. More specifically, the present invention relates to an optical method utilizing optical spectrum of the biological fluids and UV- absorbance, preferable UV-absorbance of the spent dialysate or the peritoneal liquid, and concentration calculation algorithm containing the transforming function to determine on the samples or on-line the concentration of the substances, which can be effected directly at the bed-side, real-time and which avoids the disadvantages caused by the analysis in a laboratory. The method and device determines the concentration of the substances in-vitro or on-line utilizing a measuring cuvette (cell) suitable for specified measurements.

Another object of the present invention is to provide a novel and practical optical urea and creatinine measuring method and device which determines quantitatively concentration of the forementioned water soluble small molecular weight substances in the spent dialysate. Those concentration values can be represented directly and easily on the monitor or screen printed. The novel method and device does not require any chemical disposables, and can be easily made and mass-produced providing an environment-friendly optical method. Another object of the present invention is to provide a practical optical method and device determining quantitatively concentration or removal of the middle molecules and protein bound uremic toxins in the biological fluids. The determined values can be represented directly and easily on the monitor or screen printed. The method and device does not require any chemical disposables, neither expensive separation techniques, and can be easily made and mass-produced providing an environment-friendly optical method.

A still further object of the present invention is to provide a method for assessing routine clinical monitoring in order to face risks of higher mortality in patients (e.g. in dialysis). A still further object of the present invention is to provide a novel, rapid, convenient and safe method for detecting concentration of substances in a liquid sample. The liquid sample can be directly dropped on the detecting cuvette for in-vitro measurements or sent a flowing stream of fluid through a flow-through cell for on-line monitoring. The method is suitable for household use when being applied to detect the concentration of substances in the biological fluids.

The features and advantages described herein are not all-inclusive and, in particular, many additional features and advantages will be apparent to one of ordinary skill in the art in view of the drawings, specification, and claims. Moreover, it should be noted that the language used in the specification has been principally selected for readability and instructional purposed, and not to limit the scope of the inventive subject matter. Brief description of the drawings

The present invention will be described below in the detailed description with reference to the accompanied drawings where:

Fig. 1 shows a block diagram of one embodiment of the invention.

Fig. 2 shows a block diagram of another embodiment of the invention applied for substances in a biological fluid during dialysis.

Urea

Fig. 3 shows the linear relationship between urea concentration measured by the known method (Predicted) and at the laboratory (Expected) for the "Calibration" group.

Fig. 4 shows the linear relationship between the urea concentration estimated by the new method (Predicted) and at the laboratory (Expected) for the "Calibration" group. The unique set of optical spectral components at certain wavelengths, to determine, preferable on-line, the concentration of the water soluble small molecular weight substance urea was: Urea (t) [mmol/L] = 1.00 + 6.28* A(t, 295nm) - 8.86* A(t, 320nm) - 0.529* A(t, 253nm). Fig. 5 shows the linear relationship between the urea concentration estimated by the known method (Predicted) and at the laboratory (Expected) for the "Validation" group.

Fig. 6 shows the linear relationship between the urea concentration estimated by the new method (Predicted) and at the laboratory (Expected) for the "Validation" group.

Creatinine (Crea)

Fig. 7 shows the linear relationship between creatinine concentration measured by the known method (Predicted) and at the laboratory (Expected) for the "Calibration" group. Fig. 8 shows the linear relationship between creatinine concentration measured by the new method (Predicted) and at the laboratory (Expected) for the "Calibration" group. The unique set of optical spectral components at certain wavelengths, to determine, preferable on-line, the concentration of the water soluble small molecular weight substance creatinine was: Crea (t) [mmol/L] = 31.7 + 231.2*A(t, 294nm) - 321.3* A(t, 314nm) -72.8* A(t, 283nm).

Fig. 9 shows the linear relationship between creatinine concentration estimated by the known method (Predicted) and at the laboratory (Expected) for the "Validation" group. Fig. 10 shows the linear relationship between creatinine concentration estimated by the new method (Predicted) and at the laboratory (Expected) for the "Validation" group.

Uric acid (UA)

Fig. 11 shows the linear relationship between the uric acid concentration measured by the known method (Predicted) and at the laboratory (Expected) for the "Calibration" group. Fig. 12 shows the linear relationship between uric acid concentration measured by the new A method (Predicted) and at the laboratory (Expected) for the "Calibration" group. The unique set of optical spectral components at certain wavelengths, to determine, preferable on-line, the concentration of the water soluble small molecular weight substance uric acid was: UA (t) [mmol/L] = - 3.896 + 86.51 *A(t, 298nm) - 91.21 * A(t, 312nm).

Fig. 13 shows the linear relationship between uric acid concentration measured by the new B method (Predicted) and at the laboratory (Expected) for the "Calibration" group. The unique set of optical spectral components at certain wavelengths, to determine, preferable on-line, the concentration of the water soluble small molecular weight substance uric acid was: UA (t) [mmol/L] = -2.78 - 1169* A_d(t, 307nm) - 181.3 * A_d(t, 284nm).

Fig. 14 shows the linear relationship between the uric acid concentration estimated by the known method (Predicted) and at the laboratory (Expected) for the "Validation" group. Fig. 15 shows the linear relationship between the uric acid concentration estimated by the new A method (Predicted) and at the laboratory (Expected) for the "Validation" group. Fig. 16 shows the linear relationship between the uric acid concentration estimated by the new B method (Predicted) and at the laboratory (Expected) for the "Validation" group. Fig. 17 shows a new, simplified mapping of dialysis dose and nutrition applied by using only two parameters - Kt/V and nPNA, suitable for automatic and time-efficient way to review the performed HD strategy based on the data from on-line dialysate side dialysis monitor.

Fig. 18 shows an example to estimate a parameter Lean Body Mass (LBM), suitable for automatic and time-efficient way to review the muscle mass and protein nutritional status of the HD patients based on the data from on-line dialysate dialysis monitor as comparison between the known blood based (LBM b) and the novel UV-absorbance method (LBM_uv).

Fig. 19 shows the linear relationship expressed as the correlation coefficient r between Urea, Uric Acid (UA), and B2M concentration measured in the spent dialysate by the new method (by the UV absorbance) and at the laboratory for the total material (number of samples N = 72) over the wavelength range of 190 - 380 nm.

Fig. 20 shows the linear relationship between the B2M concentration in the spent dialysate estimated by the UV absorbance at 314 nm (Predicted) and at the laboratory (Expected) for the total material. Fig. 21 shows a Bland- Altman plot of the estimated parameter eKt/V_B2M for 8 patients as the differences between eKt/Vb_B2M and eKt/Va_B2M (number of HDF sessions N = 20) plotted against mean_eKt/V (b&a)_B2M after bias correction.

Fig. 22 illustrates the dialysis dose for B2M as eKt/Va_B2M from the optical method measured in the spent dialysate plotted against eKt/Vb_B2M estimated using the blood samples (number of HDF sessions N = 20). A line of unity as a dashed line is also shown. Detailed description of the inventions

Fig. 1 shows a block diagram of one embodiment of the invention applied for determining content of the hardly or easily diffusible uremic retention solutes in the biological fluids. Another embodiment of the invention is shown in Fig. 2. The device is applied for determining the concentration of substances (uremic retention solutes) in a biological fluid during dialysis in spent dialysate 6. The device comprises spectrophotometer 7, a signal processing unit 8 for concentration calculation, a unit 9 for presenting concentration or treatment map 10 of the substance in the spent dialysate.

The block diagram of the device according to one embodiment of the invention is shown in Fig. 1. The device for measuring concentration of a hardly diffusible uremic retention solute 5 in a biological fluid 1 comprises:

an optical module 2, comprising a spectrophotometrical system, comprising a light source and a light detector;

a measuring cuvette for holding a sample of the biological fluid so that the light can be led through the sample;

a signal processing module 3 comprising a data acquisition module and a spectra processing module, and

a data representing module 4.

The light source can be either a broadband light source or a narrowband light source. If broadband light source is used, either a broadband detector with a filter can be used, or narrowband detectors. According to one embodiment, the optical module is operating in the ultra violet region (wavelength range 190-330 nm).

The measuring cuvette can be, e.g., adapted for in-vitro measurements, or designed for the on-line measurements.

According to one embodiment, the signal or spectra processing module is adapted to utilize the raw UV absorbance values or slope of the natural logarithimic UV absorbance values vs time to execute a dialysis dose calculation algorithm. According to one embodiment, the signal or spectra processing module is adapted to utilize a unique set of optical spectral components at certain wavelengths, adapted to execute a concentration calculation algorithm comprising a transforming function calculating, preferable real-time, the concentration of the uremic retention solutes in the biological fluid. The transforming function, in order to transform UV-absorbance (dimensionless) into uremic retention solute concentration in mg/L or mmol/L, has the form

y = bO + bl *Al + b2*A2 + ... + bi*Ai + b(i+l)*xl + ...+ bn*xn,

where y is a dependent variable (i.e. uremic retention solute concentration in mg/L or mmol/L), bO is the intercept, bi-s are the appropriate regression coefficients, Al - Ai-s are the measured UV-absorbances at the certain wavelengths, and xi-s are other appropriate variables.

The data representing module is adapted to execute a program for data representation and comprises or is connected to a data visualization module, e.g., a monitor, a display, or a printing device.

The data visualization module is adapted for a new, simplified mapping of dialysis dose and nutrition is applied by using only two parameters - Kt V and nPNA, suitable for automatic and time-efficient way to review the performed HD strategy based on the data from on-line dialysate side dialysis monitors.

Examples

Example 1 Concentration measurements of urea, creatinine and uric acid, in the spent dialysate

Concentration measurements of certain easily diffusible uremic retention solutes, urea, creatinine and uric acid, in the spent dialysate is given as an example of the present invention.

Subjects: A group of uremic patients on chronic thrice-weekly hemodialysis were included in the study at the Department of Dialysis and Nephrology. The dialysate flow was 500 mL/min and the blood flow varied between 245 to 350 mL/min. The type of dialysis machine used was Fresenius 4008H (Fresenius Medical Care, Germany).

Methods: The optical module consisted of a double-beam spectrophotometer (SHIMATSU UV-2401 PC, Japan) with an accuracy of ±1% on the dialysate samples taken at predetermined times during dialysis. Spectrophotometric analysis over a wavelength range of 190 - 380 nm was performed by a cuvette with an optical path length of 1 cm. The data acquisition module consisted of a PC incorporated in the spectrophotometer using UV-PC software (UV-PC personal spectrophotometer software, version 3.9 for Windows). The obtained UV-absorbance values were processed and presented by a signal processing module using a known (uniwavelength) algorithm, and the novel (multiwavelength) algorithm by a specially developed application by a concentration calculation algorithm containing the transforming function calculating the concentration of certain substance in the biological fluid. The data representing module was either the computer screen or a printer.

The dialysate samples were taken during the dialysis. Pure dialysate was collected before the start of a dialysis session, used as the reference solution, when the dialysis machine was prepared for starting and the conductivity was stable.

The concentrations of the substances such as urea, creatinine and uric acid, were determined at the clinical chemistry laboratory using standardized methods. On the basis of the results the determination coefficient R², systematic error (BIAS) and standard error (SE) were calculated for the known and new method using concentrations from the laboratory as the reference as:

N

∑«,

BIAS = -^—

where e,- is the i-th residual and N is the number of observations. The i-th residual was obtained as the difference between laboratory and optically determined parameter values for the i-th measurement.

The results (see Results) obtained by the closest existing method for determination of the amount of waste products in the dialysis liquid during dialysis treatment described in W09962574, US6666840B1 is referred here as the„known method". Those results are compared to the results by the method subject to this invention noted as the„new method". The linear relationship between urea, creatinine and uric acid concentrations, measured by the known and new method (Predicted) and at the laboratory (Expected), was first assessed using only a part of the data material, called as the "Calibration" group. After creating a model for the "Calibration" group the obtained model was applied on the rest of the material, called as the "Validation" group, not included into the model build up.

Including new patients should be the most sensitive because of the possible different combination or composition of the UV-absorbing compounds filtered from the blood into the dialysate during the dialysis may influence the model accuracy. To test a such situation only a part of the patients were included into the "Calibration" group to create a model, and after that new patients not presented before were included into the "Validation" group to validate the model.

Results

Fig. 3 and Fig. 4 show the linear relationship between urea concentration measured by the known and new method (Predicted) and at the laboratory (Expected) for the "Calibration" group.

The new method utilises an unique set of optical spectral components at certain wavelengths, to determine, preferable on-line, the concentration of the water soluble small molecular weight substance urea obtained by building up the model on the "Calibration" group. The transforming function, in order to transform UV-absorbance (dimensionless) into urea concentration [mmol/L], has the form

Urea (t) [mmol/L] = 1.00 + 6.28* A(t, 295nm) - 8.86* A(t, 320nm) - 0.529* A(t, 253nm) where Urea (t) [mmol/L] is a dependent variable urea concentration in mmol/L at the time moment t, A(t, λί nm) are the measured variables, i.e. absorbances at certain wavelengths at the time moment t multiplied by the appropriate coefficients.

Fig. 5 and Fig. 6 show the linear relationship between urea concentration measured by the known and new method (Predicted Urea) and at the laboratory (Expected Urea) for the "Validation" group including the material not included into the model build up.

Fig. 7 and Fig. 8 show the linear relationship between creatinine concentration measured by the known and new method (Predicted Crea) and at the laboratory (Expected Crea) for the "Calibration" group. The R² is increased remarkably compared to the known method.

The new method utilises an unique set of optical spectral components at certain wavelengths, to determine, preferable on-line, the concentration of the water soluble small molecular weight substance creatinine obtained by building up the model on the "Calibration" group. The transforming function, in order to transform UV-absorbance (dimensionless) into creatinine concentration [mmol/L], has the form

Crea (t) [mmol/L] = 31.7 + 231.2*A(t, 294nm) - 321.3* A(t, 314nm) -72.8* A(t, 283nm) where Crea (t) [mmol/L] is a dependent variable creatinine concentration in mmol/L), at the time moment t, A(t, λί nm) are the measured variables, i.e. absorbances at certain wavelengths at the time moment t multiplied by the appropriate coefficients.

Fig. 9 and Fig. 10 show the linear relationship between creatinine concentration estimated by the known and new method (Predicted Crea) and at the laboratory (Expected Crea) for the "Validation" group. The R² is increased remarkably utilizing the new method compared to the known method even for the "Validation" group including the material not included into the model build up.

Fig. 1 1 show the linear relationship between uric acid concentration estimated by the known method (Predicted) and at the laboratory (Expected) for the "Calibration" group. Fig. 12 and Fig. 13 show the linear relationship between uric acid concentration estimated by the new methods (A and B) (Predicted) and at the laboratory (Expected) for the "Calibration" group. The R² is increased utilizing the new method compared to the known method even for the "Calibration" group.

The new method A utilises an unique set of optical spectral components at certain wavelengths, to determine, preferable on-line, the concentration of the water soluble small molecular weight substance uric acid obtained by utilizing measured UV-absorbance values, and building up the model on the "Calibration" group. The transforming function of this method, called as "New A method", in order to transform UV-absorbance (dimensionless) into uric acid concentration [mmol/L], has the form

UA (t) [mmol/L] = - 3.896 + 86.51 *A(t, 298nm) - 91.21 * A(t, 312nm)

where UA (t) [mmol/L] is a dependent variable uric acid concentration in mmol/L at the time moment t, A(t, λί nm) are the measured variables, i.e. absorbances at certain wavelengths at the time moment t multiplied by the appropriate coefficients.

Additionally, the new method B utilises an unique set of optical spectral components at certain wavelengths, to determine, preferable on-line, the concentration of the water soluble small molecular weight substance uric acid obtained by utilizing the processed UV- absorbance values by Sawitsky-Golay method, and building up the model on the "Calibration" group. The transforming function of this method, called as "New B method", in order to transform UV-absorbance (dimensionless) into uric acid concentration [mmol/L], has the form

UA (t) [mmol/L] = -2.78 - 1 169* A_d(t, 307nm) - 181.3* A_d(t, 284nm)

where UA (t) [mmol/L] is a dependent variable uric acid concentration in mmol/L at the time moment t, A_d(t, λί nm) are the variables (measured and the processed UV-absorbance values by Sawitsky-Golay method), i.e. processed absorbances at certain wavelengths at the time moment t multiplied by the appropriate coefficients.

Fig. 14 show the linear relationship between uric acid concentration estimated by the known method (Predicted) and at the laboratory (Expected) for the "Validation" group. Fig. 15 and Fig. 16 show the linear relationship between uric acid concentration estimated by the new methods (A and B) (Predicted) and at the laboratory (Expected) for the "Validation" group. The R² is increased remarkably utilizing the new method compared to the known method even for the "Validation" group including the material not included into the model build up.

As seen from the Tables 1-3 determination of urea, creatinine and uric acid concentrations can be done much more precisely applying the "New method". The R is increased, and validation SE is decreased compared to the "Known method".

Table 3: Summary results for the different methods to measure concentration of the acid.

This means that utilizing the new methods the concentrations of urea, creatinine and uric acid can be predicted more accurately in terms of BIAS and SE.

Fig. 17 shows a new, simplified mapping of dialysis dose and nutrition applied by using only two parameters - Kt/V and nPNA, suitable for automatic and time-efficient way to review the performed HD strategy based on the data from on-line dialysate side dialysis monitor, utilizing the urea concentration determination method described above. Fig. 18 shows an example to estimate a parameter Lean Body Mass (LBM), suitable for automatic and time-efficient way to review the muscle mass and protein nutritional status of the HD patients based on the data from on-line dialysate dialysis monitor as comparison between the known blood based (LBM b) and the novel UV-absorbance method (LBM_uv), utilizing the creatinine concentration determination method described above. Although this invention is described with respect to a set of aspects and embodiments, modifications thereto will be apparent to those skilled in the art. The foregoing description of the embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of this disclosure. It is intended that the scope of the invention be limited not by this detailed description, but rather by the claims appended hereto.

Example 2 Concentration measurement of a hardly diffusible middle molecule beta2- microglobulin (B2M)

Concentration measurement of a certain hardly diffusible uremic retention solute, a middle molecule beta2-microglobulin (B2M), in the spent dialysate, is given as an example of the present invention.

Subjects: Eight uremic patients, one female and seven males, were included in the study. All patients were on chronic three -weekly on-line HemoDiaFiltration (ol-HDF) at the Department of Nephrology, University Hospital of Linkoping, Sweden. The dialysis machine used was a Fresenius 5008 (Fresenius Medical Care, Germany). The dialyzers used were in all treatments FX 800 (Fresenius Medical Care, Germany), with an effective membrane area of 1.8 m², with an ultra filtration coefficient of 63 ml/h mmHg. The duration of the ol-HDF treatments varied between 180 to 270 minutes, the dialysate flow was 500 mL/min, the blood flow varied between 280-350 mL/min. All patients were dialyzed via artery-venous fistulas using a "two-needle" system. The auto sub system mode for calculation of the on-line prepared substitution volume varied between 12.2 to 29.7 liters per session.

Sampling: Samples from the drain tube were taken at (min) 9 and at the end of ol-HDF session. One sample was taken from the dialysate/ultrafiltrate collection tank after careful stirring and weighing was performed. If a self-test of the dialysis machine occurred during the planned sampling time, the sample was taken when the UV-absorbance curve reached baseline level again which occurred within 2-3 minutes. Pure dialysate was collected before the start of a dialysis session, used as the reference solution, when the dialysis machine was prepared for starting and the conductivity was stable.

Determination of concentrations of B2M in the spent dialysate/ultrafiltrate was performed at the Chemical Laboratory, University Hospital in Linkoping.

Results: Fig. 19 illustrates the examples of shows the linear relationship expressed as the correlation coefficient r between Urea, Uric Acid (UA), and B2M concentrations measured in the spent dialysate by the new method (by the UV absorbance) and at the laboratory for the total material (number of samples N = 72) over the wavelength range of 190 - 400 nm. Some distinctive correlation maxima at specific regions for different uremic solutes are clearly seen. The linear relationship analysis for B2M, utilizing UV absorbance values at the wavelength which yielded the strongest correlation (314 nm) and the concentrations from the laboratory. This led to a specific model which enabled transform the optical measurements into the B2M concentration values. The function, in order to transform UV-absorbance (dimensionless) into the B2M concentration in mg/L, had the form:

B2M (t) [mg/L] = 8.13* A(t, 314 nm).

The determined values of B2M concentrations in the spent dialysate by the the new method, obtained by the specific model utilizing the UV absorbance at 314 nm (Predicted B2M) have strong linear relationship between the B2M concentrations estimated at the laboratory (Expected B2M) for the total material (Fig 20).

Table 4 summarises all results about the B2M concentrations as mean and standard deviation values (Mean +/- SD) from the standardised methods (Lab) and from the new method (UV). The linear correlation coefficient (r) and the R-squared value (R ) between the B2M concentration from the optical method and concentration measured at the laboratory, the accuracy (BIAS) and precision (SE) for the different methods to measure concentration of B2M, are also given.

Table 4: Summary results about the concentration mean and standard deviation values (Mean +/- SD) from the standardised methods (Lab) and new method (UV), linear correlation coefficient (r) and the R-squared value (R ) between the uremic toxins concentration from the optical method and concentration measured at the laboratory, the accuracy (BIAS) and precision (SE) for the different methods to measure concentration of

B2M.

SE rmg/Ll 0.37

As seen from the Table 4 determination of B2M concentration can be done with satisfactory accuracy and precision applying the novel method.

As an example for clinical application, the removal of B2M from the optical measurements is estimated below to calculate the dialysis dose for B2M, being representative in its kinetic behavior of other MM and peptides of similar size.

The dialysis dose for the B2M from blood, spKt/Vb_B2M and eKt/Vb_B2M, can be calculated using the pre- and post- dialysis blood B2M concentrations (C₀ and C_t). The single pool volume Kt/V, spKt/Vb_B2M was calculated according to the formula proposed by Casino et al 2010 [1 1], as

where UF is the total ultrafiltration in kg and W is the patient's dry body weight in kg. The equilibrated Kt/V, eKt/Vb_B2M, taking account post-dialysis B2M rebound, was obtained according to the formula proposed by Tattersall et al 2007, as:

eKt/Vb _ B2M = spKt/V_p2m * T_d /(T_d + 11 θ) (4 where T_d is the dialysis session length.

For determination of the dialysis dose for B2M from the optical method, instead of the pre- and post-dialysis blood B2M concentrations, the UV-absorbance value in the beginning, A₀ (9 min dialysate sample) and at the end of dialysis, A_t , were utilized at the wavelength which yielded the strongest correlation between B2M concentrations measured in the spent dialysate by the new method (by the UV absorbance) and at the laboratory (314 nm). The single pool volume Kt/V from the absorbance measurements, spKt/V a_B2M was calculated as

The equilibrated Kt/V from the absorbance measurements, eKt/Va_B2M was calculated according to equation 4.

Table 5 summarises all results about the dialysis dose for the B2M as eKt/V_B2M calculated using the pre- and post- dialysis blood B2M concentrations and the absorbance values from totally 20 HDF sessions. The linear correlation coefficient (r) and the R-squared value (R2) between the dialysis dose for B2M from the optical method and dialysis dose for B2M from the blood concentrations are given. The accuracy (BIAS) and precision (SE) for the optical method was calculated using dialysis dose for the B2M from blood as reference after bias correction.

Table 5: Summary of dialysis dose as eKt/V_B2M, calculated by the TDC and the absorbance values on the tank dialysate sample (UV) , the linear correlation coefficient (r) and the R-squared value (R ) between the dialysis dose for B2M from the optical method and from the blood concentrations, the accuracy (BIAS) and precision (SE) for the optical method.

Fig. 21 presents a comparison as a Bland- Altaian plot of the estimated parameter eKt/V_B2M for all 8 patients as the differences between eKt/Vb_B2M and eKt Va_B2M (number of HDF sessions N = 20) plotted against mean_eKt/V(b&a)_B2M after bias correction. Fig. 22 illustrates the dialysis dose for B2M as eKt/Va_B2M from the optical method measured in the spent dialysate plotted against eKt/Vb_B2M estimated using the blood samples (number of HDF sessions N = 20). A line of unity as a dashed line is also shown.

The results show good agreement between the dialysis dose estimated for B2M from the blood samples and from the spent dialysate samples by the UV-absorbance.

As the third example for clinical application, the concentration of B2M from optical measurements is utilized below to calculate the total removed B2M, TR_B2M.

The TR B2M from TDC (reference) was calculated as B2M concentration in the tank at the end of dialysis multiplied by collected weight [kg], assuming that 1kg = 1L of the dialysate. Similarly, TR B2M from the UV-absorbance was calculated as B2M concentration in the tank at the end of dialysis estimated by UV-absorbance utilising the transforming function, and multiplied by collected weight [kg], assuming that lkg = 1L of the dialysate.

Table 6 summarises all results about the dialysis dose for the B2M as the total removed B2M, TR_B2M, calculated using the B2M concentrations in the tank at the end of dialysis estimated at the laboratory (TDC), and by the UV-absorbance (UV) from totally 20 HDF sessions. The linear correlation coefficient (r) and the R-squared value (R2) between the dialysis dose for B2M from the optical method and dialysis dose for B2M from the blood concentrations are given. The accuracy (BIAS) and precision (SE) for the optical method was calculated using dialysis dose for the B2M from blood as reference after bias correction.

Table 6: Summary of dialysis dose as TR B2M, calculated using using the B2M concentrations in the tank at the end of dialysis estimated at the laboratory (TDC) and by the UV-absorbance (UV), the linear correlation coefficient (r) and the R-squared value (R²) between the dialysis dose for B2M from the optical method and from the blood concentrations, the accuracy (BIAS) and precision (SE) for the optical method.

As seen from the Table 6 determination of TR B2M can be done with satisfactory accuracy and precision applying the novel method.

Although this invention is described with respect to a set of aspects and embodiments, modifications thereto will be apparent to those skilled in the art. The foregoing description of the embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of this disclosure. It is intended that the scope of the invention be limited not by this detailed description, but rather by the claims appended hereto. Patent references:

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Claims

1. A method for measuring and monitoring concentration of substances in a biological fluid, the method comprising the following subsequent steps of:

a) introducing the sample or flowing stream of the biological fluid into a cuvette; b) applying light from light source with predetermined wavelengths to the sample of the biological fluid and recording an optical spectrum of said sample by the light detector;

c) using the transforming function including a unique set of optical spectral components at certain wavelengths for calculating, preferable in real-time, the concentration of the substances in said biological fluid.

2. The method as in claim 1 , wherein after step c) the raw UV absorbance values or slope of the natural logarithmic UV absorbance values versus time are used to execute a dialysis dose calculation algorithm.

3. The method as in claim 1, wherein flowing stream of the biological fluid is introduced through flow-cuvette.

4. The method as in claim 1 , wherein substances in a biological fluid are hardly or easily diffusible uremic retention solutes.

5. The method as in claim 4, wherein the hardly diffusible uremic retention solute is beta2- microglobulin.

6. The method as in claim 4, wherein the easily diffusible uremic retention solutes are urea, creatinine or uric acid.

7. The method as in claim 1 , wherein the biological fluid comprising said substance is exposed in the cuvette to the wavelength induced by the light source in the ultra violet region.

8. The method as in claim 7, wherein the biological fluid comprising said substance is exposed to the wavelength induced by the light source from 180-400 nm.

9. The method as in claim 7, wherein the light source and the light detector are operating in the wavelength range 300-330 nm, suitable for beta2-microglobulin measurements.

10. The method as in claim 7, wherein the light source and the light detector are operating in the wavelength range 180-310 nm, suitable for urea, creatinine or uric acid.

11. The method as in claim 1, wherein the calculating the concentration of the substances in a biological fluid comprises executing a transforming function according to the formula y = bO + bl *xl + b2*x2 + b3*x3 + ...+ bn*xn, where y is a concentration of the substance in a biological fluid in mmol/L, xi-s are the measured absorbances or other important parameters in the wavelengths range 180-400 nm, and bi-s are the appropriate coefficients.

12. The method as in claim 2, wherein the signal dialysis dose calculation algorithm utilises directly raw UV- absorbance values or slope of the natural logarithimic UV absorbance values vs time.

13. The method as in claim 1 , wherein the sample of the biological fluid comprising said substances is dropped onto in- vitro cuvette.

14. The method as in claim 1, wherein the sample of the biological fluid comprising said substances is obtained and introduced into cuvette in home conditions.

15. The method as in claim 1, wherein the concentrations of the substances in a biological fluid are outputted to a display device or to a printer.

16. A method of monitoring of a patient by monitoring removal of the substances in a biological fluid during dialysis and by measuring concentration of said substances in patient's biological fluid, comprising:

a) introducing a flow of patients biological fluid into flow-cuvette;

b) applying light with predetermined wavelengths to the sample and recording an optical spectrum of the sample;

c) using the transforming function including a unique set of optical spectral components at certain wavelengths for calculating, preferably in real-time, the concentration of the substances in a biological fluid in the sample from the patient's biological fluid.

17. The method as in claim 16, wherein the concentration data measured during the monitoring of said substance are recorded and stored in a memory device.

18. The method as in claim 16, comprising visualization module adapted for a novel mapping of dialysis dose and nutrition by using only two parameters - Kt/V and nPNA for urea, suitable for automatic and time-efficient way to review the performed HD strategy based on the data from on-line dialysate side dialysis monitors.

19. The method as in claim 16, comprising calculation of a parameter Lean Body Mass (LBM) for creatinin, suitable for automatic and time-efficient way to review the muscle mass and protein nutritional status of the HD patients based on the said substance concentration determination method.

20. The method as in claim 16, comprising a comparisation of the substance concentration level in the biological fluid with predetermined limits of the concentration and generating an alarm signal, if the concentration does not fall between predetermined limits.

21. A device for measuring and monitoring concentration of substances or for monitoring removal of substances in a biological fluid utilizing UV-absorbance measurements during dialysis, the device comprising:

- a measuring cuvette for holding a sample or passing through the biological fluid; - an optical module comprising a spectrophotometrical system, comprising a light source for introducing light into the sample, and a light detector for receiving light from the sample;

- a signal processing module comprising a data acquisition module, a spectra processing module and a data representing module, the data representing module adapted to execute a program for data representation and comprising a data visualization module.

22. The device as in claim 21 wherein the substances in a biological fluid introduced into a measuring cuvette are hardly or easily diffusible uremic retention solutes.

23. The device as in claim 21, the light source and the light detector are operating in wavelength range 180-400 ran.

24. The device as in claim 23, the light source and the light detector are operating in the wavelength range 300-330 nm, suitable for beta2-microglobulin measurements.

25. The device as in claim 21 , wherein the light source is a broadband light source.

26. The device as in claim 21 , wherein the optical module comprises a filter and a broadband detector.

27. The device as in claim 21, wherein the optical module comprises narrowband detectors.

28. The device as in claim 21, wherein the optical module comprises a set of narrowband light sources and a broadband detector.

29. The device as in claim 21, wherein the cuvette is a flow-cuvette for receiving a flowing stream of the spent dialysate.

30. The device as in claim 29, wherein the cuvette is adapted for in-vitro measurements.

31. The device as in claim 21, wherein the signal processing module is adapted to execute a concentration calculation algorithm containing the transforming function calculating the concentration of substances in the biological fluid according to the formula y = bO + bl *xl + b2*x2 + b3*x3 + ...+ bn*xn, where y is a hardly or easily diffusible uremic retention solute concentration in mmol/L, xi-s are the measured absorbances or other important parameters in the wavelengths range 190-400 nm, and bi-s are the appropriate coefficients.

32. The device as in claim 21, wherein the signal processing module is adapted to execute a concentration calculation algorithm containing the transforming function, including a unique set of optical spectral components at certain wavelengths, whereas the transforming function, in order to transform UV-absorbance (dimensionless) into the concentration of a substance in a biological fluid [mmol/L], has the formula y = bO + bl *xl + b2*x2 + b3*x3 + ...+ bn*xn, where y is the solute concentration in mmol/L, xi-s are the measured absorbances or other important parameters, and bi-s are the appropriate coefficients.

33. The device as in claim 21, wherein the signal processing module is adapted to execute a dialysis dose calculation algorithm utilizing directly raw UV- absorbance values or slope of the natural logarithimic UV absorbance values vs time.