**4.1 Uremic toxins**

Uremic toxins used to be divided into three different molecule groups, that is small, water soluble, middle and protein-bound [72], but it needs still to work out their relation to dialysis efficacy and what roles different molecules will have in the development of uremia and which dialysis techniques are best at reducing the elevated levels of these [73]. Proposal for a new classification system of uremic solutes rationale has been made [74]. Declined uremic toxin clearance due to low GFR is not the only cause of toxin accumulation in kidney failure. Excessive production of cytokines and soluble receptors due to local tissue inflammation is a major contributor to the middle molecule accumulation [75], and gut dysbiosis generates a broad spectrum of uremic toxins [76]. Thus, a broader view of uremic solutes that goes beyond simply retention with poor GFR is needed. Recent data regarding the origin of uremic toxins, and the new development of HD methods and new membranes with the ability to clear uremic toxins with specific characteristics, or by using drugs/molecules to facilitate the shift from bound fraction to free fraction [77], have led to propose a new classification beyond the classic physicochemical classification. A study by Vanholder et al. (2018) presents a ranking score list of uremic toxins with known toxicity according to experimental and clinical studies [78], **Table 1** shows the highest- and second-highest evidence-scored uremic toxins.

The uremic toxins with the optical monitoring capacity referred to further in this chapter are marked in underline. AGEs and IAA are in italics as the potential targets for the optical monitoring in the future, **Table 1**.


**Table 1.**

*Uremic toxins with the highest toxicity evidence score (modified from Vanholder et al. 2018) [78].*

### *4.1.1 Optical intradialytic monitoring of small water-soluble molecules' removal*

Uric acid, like urea, a representative molecule from the small group of uremic toxins, has been shown to have a high absorption of UV in the wavelength region 280– 310 nm (also a peak around 230–240 nm), **Figure 5a**. As a consequence, we have been able to show that it is possible to estimate the total removal of uric acid during dialysis

#### **Figure 5.**

*(a) The molar absorptivity for four solutes, β2M, uric acid, creatinine and urea in 24 HDF sessions. (b) An example of the regression line between concentration of uric acid and UV-absorbance [Figure 5a, reprinted from [86]].*

#### *Optical Online Monitoring of Uremic Toxins beyond Urea DOI: http://dx.doi.org/10.5772/intechopen.110080*

[79, 80] and a multi-wavelength and processed signal approach can provide even more accurate results [81]. **Figure 5b** shows an example of the best-fit regression equation of uric acid vs. UV absorbance in spent dialysate at wavelength 285 nm during four dialysis sessions in the same patient, showing a high correlation of r = 0.99.

Earlier research has demonstrated potential of intradialytic optical monitoring to estimate the removal of low molecular weight uremic solutes other than uric acid as urea [53] and creatinine [82] with their clinical implications. Furthermore, optical monitoring of low molecule weight uremic solutes removal by HD, assessed via the marker molecule urea-related dialysis adequacy parameters, has become a worldwide practice [55, 56, 83]. A possibility for optical monitoring of phosphate and calcium elimination during dialysis has also been presented [84, 85].

#### *4.1.2 Optical intradialytic monitoring of middle molecules' removal*

Using UV absorbance alone to estimate β2M does not appear to be optimal even a high correlation between UV absorbance and β2M in spent dialysate can be achieved for HDF but not for HD [86]. Instead, fluorescence spectra could be a better alternative [87] as it is possible to detect the fluorescence of advanced glycation end products (AGE) modified β2M in spent dialysate [88, 89]. However, the measuring system needs high selectivity and sensitivity for detection due to low contribution of AGE modified β2M to overall fluorescence [89]. The best correlation between the fluorescence of spent dialysate and the concentration of β2M in spent dialysate was found in the wavelength region Ex350–370/Em500–555 nm, with the coefficient of determination R2 up to 0.859, **Figure 6a**.

A multiwavelength fluorescence approach can yield a high correlation of up to 0.958 between laboratory and optically estimated β2M concentrations in spent dialysate. The main contributors to the optical signal of the middle molecule (MM) fraction were provisionally identified as tryptophan (Trp) in small peptides and proteins and AGEs [90]. **Figure 6b** visualize the good agreement between β2M concentrations analyzed in laboratory vs. those predicted by an optical model during 29 four-hour HDF sessions.

#### *4.1.3 Optical intradialytic monitoring of protein-bound molecules' removal*

Recent studies have presented that quantification of indoxyl sulphate (IS) in the spent dialysate using fluorescence spectra is possible [91, 92]. **Figure 7** shows an example of a HPLC chromatogram of a spent dialysate sample taken 10 min after the start of HD, where the fluorescence was recorded at Ex: 280 nm and Em: 360 nm. In total, 12 clearly resolved chromatographic peaks of fluorophoric compounds were detected in most (82%) of the spent dialysate samples during the HPLC analysis collected at different time moments in the dialysis [92]. Of these, five peaks had a major importance in all samples (peaks no 6, 8, 9, 11 and 12), and six of these 12 peaks were identified as Trp and their metabolites of indole derivates: indoxyl glucuronide (IGluc), IS, 5-hydroxy-indole-3-acetic acid, indole acetyl glutamine (IaG) and indole acetic acid (IAA) [92]. IS is one of the main fluorophores in these measuring conditions (**Figure 7**, peak 9) [92]. This is in agreement with the earlier studies, where IS has been found as a main contributor to the fluorescence in uremic fluids [93, 94].

#### **Figure 6.**

*a. Wavelength dependence of the correlation between fluorescence intensity and concentration of β2M in spent dialysate for HDF modalities (N = 375). b. Time-series of changing β2M concentration (mean SD) in the spent dialysate during HDF dialysis sessions (N = 29) for patients of the validation set [Reprinted from [90]].*
