**3. Optical dialysis online monitoring**

Two optical techniques have made progress toward clinical use over the past ten years – namely the ultraviolet (UV) absorbance and near infrared (NIR) techniques. The experimental results indicate very good correlation between UV absorbance and several small solutes such as urea, creatinine and uric acid in the spent dialysate and in the blood for every individual treatment at a fixed wavelength of 285 nm (Fridolin, 2002). Good correlation between UV absorbance and a small removed waste solute such as urea enables the determination of Kt/V for urea (Fridolin, 2003). The NIR method can measure urea directly using signal processing of the raw NIR spectra (Cho, 2008; Eddy, 2001; Eddy, 2003). To implement NIR technology as a simple and robust sensor based on Fourier transform near-infrared (FT-NIR) spectrometer or acousto optical tunable filter (AOFT) based

recognition of signs of intolerance and allows for early intervention. Despite the first online monitors of dialysis doses being available today, it is essential to monitor the patient's condition during the dialysis session and, if necessary, change some conditions of dialysis treatment (e.g. time) more specifically considering uremic toxins with various characteristics

The aim of this chapter is to describe and assess performance of optical dialysis adequacy monitoring technique related to the removal of uremic retention solutes during dialysis. The assessment is based on the high performance liquid chromatography (HPLC) profiles of the serum and the spent dialysate connected to the origin of the cumulative and integrated UV absorbance arising from the contribution of uremic retention solutes - chromophores, among

Dialysis adequacy means providing a sufficient amount of dialysis treatment to maintain a uremic patient in the best condition. The goal of qualitatively treated dialysis is to prevent

Some recent studies are reviewed suggesting that uremic toxins are involved in the progression of renal failure and are at least partially removed by hemodialysis (Lesaffer, 2000; Eloot, 2008; Vanholder, 2003). The efficiency of a dialysis session has been estimated through concentration of uremic toxins measured before and after the dialysis session. A

Two methods are generally used to assess dialysis adequacy: URR and Kt/V. The urea reduction ratio (URR) is based on tests of blood urea, by measuring the levels before and after treatment to show how much has been removed. The Kt/V is mathematically related to the URR and can be derived from it (NKF-DOQI, 2006). In a simplified model of urea removal from a fixed volume with no urea generation, Kt/V is related to URR as follows:

In general, this method of dialysis adequacy is based on pre- and post-dialysis measurements of urea concentration. Online methods are considered to be more accurate than methods based on pre- and post-dialysis urea concentrations and have been found to be better suited to clinical routines. Online monitoring of the dialysis dose has been suggested as a valuable tool in ensuring adequate dialysis prescription (Locatelli, 2005).

Two optical techniques have made progress toward clinical use over the past ten years – namely the ultraviolet (UV) absorbance and near infrared (NIR) techniques. The experimental results indicate very good correlation between UV absorbance and several small solutes such as urea, creatinine and uric acid in the spent dialysate and in the blood for every individual treatment at a fixed wavelength of 285 nm (Fridolin, 2002). Good correlation between UV absorbance and a small removed waste solute such as urea enables the determination of Kt/V for urea (Fridolin, 2003). The NIR method can measure urea directly using signal processing of the raw NIR spectra (Cho, 2008; Eddy, 2001; Eddy, 2003). To implement NIR technology as a simple and robust sensor based on Fourier transform near-infrared (FT-NIR) spectrometer or acousto optical tunable filter (AOFT) based

Kt/V = -ln (1 – URR) (1)

classic marker of the dialysis dose and adequacy is a small molecule urea.

and elimination profiles.

them probably several uremic toxins.

complications due to uremic toxicity.

**3. Optical dialysis online monitoring** 

**2. Dialysis adequacy and online monitoring** 

spectrometer is far more complicated because of the underlying optical principle, in which interference or diffraction is utilised (Jensen, 2002; Cho, 2008). The UV method is more straightforward and is not as demanding considering the source and detector characteristics and other technological modules. The UV method has recently been commercialised as a monitoring tool for dialysis dose in terms of the urea-based parameters, Kt/V and URR. Validation studies of the system have shown that the results with UV technology are indistinguishable from blood based Kt/V (Castellarnau, 2010).

An optical dialysis online monitoring system utilising UV absorbance has been developed for the continuous monitoring of the toxins eliminated by the spent dialysate. This system represents a spectrophotometer connected to the fluid outlet of the dialysis machine. All spent dialysate passes through a specially designed optical cuvette. The transmitted light intensity of the spent dialysate is measured. All substances – chromophores – absorbing the UV-radiation at a particular wavelength, give the total online UV absorbance curve. The schematic clinical set-up of online monitoring experiments is shown in Figure 1.

Fig. 1. Clinical experimental set-up and sample analysis

The obtained UV absorbance values are processed and presented on the computer screen by a PC incorporated in the spectrophotometer using special software. The absorbance A of a solution, obtained by the spectrophotometer using the pure dialysate as the reference solution, is determined as:

$$A = \log \frac{I\_r}{I\_{r \leftrightarrow s}} \tag{2}$$

where Ir is the intensity of transmitted light through the reference solution (pure dialysate) and Ir+s is the summated intensity of transmitted light through the reference solution containing the solutions under study (pure dialysate + waste products from the blood). The absorbance is measured in arbitrary units (a.u.). A sampling frequency at two samples per minute is usually sufficient, but can be much higher (e.g. if a more detailed curve is desired or noise reduction is necessary).

Optical Dialysis Adequacy Monitoring:

chromatography.

size of non-polar groups.

and data-processing.

**4. HPLC as an analysis tool for biological samples** 

Fig. 3. Schematic reversed-phase HPLC method principle

Small Uremic Toxins and Contribution to UV-Absorbance Studied by HPLC 147

High performance liquid chromatography (HPLC) is a technique of analytical chemistry which can separate and identify the components of a mixture of different chemical compounds in liquid solution. The reversed-phase HPLC technique is the most commonly used form of HPLC. This method is recommended as a sensitive, accurate and reproducible tool for qualitative and quantitative analysis of aqueous samples (Vanholder, 2001). Furthermore, the use of ambient temperature in reversed-phase columns makes it possible to investigate the many non-volatile or thermally unstable compounds commonly found in biological samples. The principle involved in HPLC testing enables the separation of compounds in a mixture more efficiently and faster than that of traditional column

In general, the HPLC system consists of two essential components – a stationary phase and a mobile phase. The stationary phase is a column packed with small solid sorbent particles and where the separation of different compounds takes place. The mobile phase is a flowing liquid (solvent) that transports the compounds from the sample through the stationary phase. Thus, the compounds of the mixture travel at different rates due to their relative affinities with the solvent and stationary phase. Separation of compounds in the stationary phase occurs with slight differences in chemical properties, such as chemical polarity and

Figure 3 is a simple block diagram illustrating the main components of a modern HPLC. A system consists of several units: pumping, sample-injection, separation (column), detection

The results of chromatographic analysis are known as chromatograms, where the signal intensity from the detector is recorded on the time axis. For HPLC there are several different detection methods; the most popular are optical. An ultraviolet-visual light (UV-VIS) absorption detector is the main optical detection method. This detector is effective in the detection of components with an absorption wavelength of 400 nm or less in the ultraviolet

The examples of two online signals measured at 280 nm from two different dialysis treatments are shown in Figure 2. The UV absorbance is higher at the beginning of treatment because of the high concentration of metabolic waste products in the body fluids. When the waste products are removed from the blood the UV absorbance decreases during the dialysis session. The times at which the blood and dialysate samples were collected are also shown in this figure. Blood samples were drawn before the start of dialysis treatment (Bstart) and immediately after the treatment (Bend). Dialysate samples were taken 10 minutes after the start of the dialysis session (Dstart) and at the end of the treatment (Dend) (210 or 240 minutes).

Fig. 2. Example of two different online UV absorbance measurements (at 280 nm). Time points when the samples were taken for later analysis are as follows: Dstart - dialysate sample collected 10 minutes after start of hemodialysis, Dend- dialysate sample collected at the end of hemodialysis, Bstart – blood sample collected before dialysis session, Bend – blood sample collected at the end of hemodialysis

This figure presents a classic picture of UV absorbance signals obtained by the optical online dialysis dose monitor, which can be used for estimation of uremic solutes removal. As seen from Figure 2, the UV absorbance curves are somewhat different. The exponential decrease of the UV absorbance curve represents the elimination rate of the all UV-absorbing compounds – chromophores – which varies from patient to patient and from treatment to treatment. Good correlation between the UV absorbance measured in the spent dialysate and the concentration of several uremic retention solutes, both in the spent dialysate and in the blood of the dialysis patients, has been previously shown (Fridolin, 2002). For these reasons the origin of the cumulative and integrated UV absorbance arising from the contribution of uremic retention solutes, among them probably several uremic toxins, should be investigated.
