**6. Discussion**

From the investigations presented in this chapter we can conclude the following: (i) UV absorbance decreases during the dialysis session as the waste products are removed; (ii) a number of higher prevalent peaks can be detected in the HPLC profiles; (iii) some lowmolecular-weight uremic solutes were identified from the HPLC profiles contributing to UV absorbance, and the main solute responsible for the UV absorbance is uric acid; (iv) a difference between HPLC profiles on wavelengths of 254 and 280 nm was found; (v) a higher number of detected HPLC peaks in the serum comparing to spent dialysate has been detected; (vi) the low flux and high flux membranes showed similar RR for all studied uremic solutes; and (vii) the classic dialysis adequacy marker urea and the uremic solute uric acid have good correlation with uremic retention solute elimination forming the UV absorbance curve.

Figures 2 and 5 show how the concentration of the uremic retention solutes – chromophores – decreases during a dialysis session in spent dialysate. UV absorbance as well as the height of the HPLC peaks are higher at the beginning of the treatment because of the high concentration of metabolic waste products in the body fluids and UV absorbance is lower at the end of the dialysis session. This demonstrates the possibility of following a single dialysis session continuously and to monitor deviations during treatment using a UV absorbance online monitor. This enables us to estimate the dialysis efficiency and adjust the treatment settings if needed.

The RR of urea and uric acid and the RR of all HPLC peaks at 280 nm are > 60%, while the RR of all HPLC peaks at 254 nm, of online UV absorbance measurement and creatinine are < 60%. Interestingly, the removal of urea and uric acid was statistically undifferentiated (p > 0.05). Figure 8 shows that the RR of serum creatinine is statistically different (p < 0.05) from the RR of serum urea and uric acid concentrations and from the RR of all HPLC peaks in the serum (at 280 nm), but not different from that of 254 nm. The RR of online measurement is comparable with the RR of creatinine and of all HPLC peaks at 254 nm and lower than the RR of urea, uric acid and all HPLC peaks at 280 nm (p < 0.05). At the same time, the RR of serum urea and uric acid are not statistically different; neither are they different from the RR

The correlation coefficients between RR UV absorbance at 254 nm and at 280 nm from the total area of the HPLC peaks, from online monitoring at 280 nm and RR for certain substances with different molecular weights in spent dialysate and serum are shown in

RR of all HPLC peaks, 254 nm 0.755 0.811 0.812 RR of all HPLC peaks, 280 nm 0.933 0.881 0.926 Online UV absorbance, 280 nm 0.890 0.873 0.888 Table 2. Pearson correlation coefficients between RR of all HPLC peaks (254 nm and 280 nm), online absorbance at 280 nm and RR of uric acid, urea and creatinine in serum. The

A high correlation for uric acid, urea and creatinine was obtained in both spent dialysate and serum. Some differences regarding 254 nm and 280 nm can be observed. However, urea

From the investigations presented in this chapter we can conclude the following: (i) UV absorbance decreases during the dialysis session as the waste products are removed; (ii) a number of higher prevalent peaks can be detected in the HPLC profiles; (iii) some lowmolecular-weight uremic solutes were identified from the HPLC profiles contributing to UV absorbance, and the main solute responsible for the UV absorbance is uric acid; (iv) a difference between HPLC profiles on wavelengths of 254 and 280 nm was found; (v) a higher number of detected HPLC peaks in the serum comparing to spent dialysate has been detected; (vi) the low flux and high flux membranes showed similar RR for all studied uremic solutes; and (vii) the classic dialysis adequacy marker urea and the uremic solute uric acid have good

correlation with uremic retention solute elimination forming the UV absorbance curve.

Figures 2 and 5 show how the concentration of the uremic retention solutes – chromophores – decreases during a dialysis session in spent dialysate. UV absorbance as well as the height of the HPLC peaks are higher at the beginning of the treatment because of the high concentration of metabolic waste products in the body fluids and UV absorbance is lower at the end of the dialysis session. This demonstrates the possibility of following a single dialysis session continuously and to monitor deviations during treatment using a UV absorbance online monitor. This enables us to estimate the dialysis efficiency and adjust the

Uric acid Urea Creatinine

of all HPLC peaks at 254 nm and 280 nm (p < 0.05).

significance level of the results is P<0.01.

does not represent as good a correlation as uric acid.

Table 2.

**6. Discussion** 

treatment settings if needed.

A number of higher prevalent HPLC peaks representing chromophores can be observed (Figure 5 and 6). This indicates that there is a group of compounds, among them several uremic toxins, which are the main cause of cumulative and integrated UV absorbance. The 10 main peaks formed app. 80-90% of the total area of all HPLC peaks; some of these are small molecular weight uremic toxins such as uric acid, creatinine, hippuric acid and indoxyl sulphate. The variations in the number of HPLC peaks depending on hemodialysis treatments and patients have been demonstrated in earlier studies (Schoots, 1982; Vanholder, 1992). The difference between two dialysis sessions may arise as shown in Figure 2, because of the different composition and removal of the uremic retention solutes contained in spent dialysate. When comparing the HPLC profiles of the spent dialysate in Figure 5 and those of the serum in Figure 6, more peaks are detected in the serum. Thus, not all solutes in serum are transported to dialysate and removed through the semi-permeable membrane.

The number of detected HPLC peaks at 254 nm and 280 nm is also demonstrated in Figure 6 and 7. The difference in the number of detected HPLC peaks on the wavelengths of 254 nm and 280 nm arises due to the characteristic absorbing spectra of the UV chromophores. The absorption of many components is higher on the wavelength of 254 nm than 280 nm. This confirms the results obtained via the spectrophotometric analysis in this UV region (Fridolin, 2003). However, the studies of relations between UV and small water soluble molecules such as urea and uric acid indicated that the wavelength of 280 nm may be preferred for online measurements when small water soluble molecules should be estimated. On this wavelength a relatively strong linear relationship exists between UV absorbance and concentrations of urea, creatinine and uric acid (Fridolin, 2002; Uhlin, 2003). While the contribution of uric acid forms a considerable part of the total area of HPLC peaks, uric acid plays an important role in online UV absorbance dialysis dose monitoring. Interestingly, the removal of urea and uric acid was statistically undifferentiated (p > 0.05). This information gives us alternative possibilities to use other components and methods to monitor urea reduction (URR) in a single hemodialysis session.

Additionally, the low flux and high flux membranes showed no different removal of the studied small molecule uremic toxins as presented in earlier studies (Lesaffer, 2000). In this study it was found that the cellulose triacetate and polysulphone HF membranes removed similarly classical markers and protein-bound liphophilic solutes as an LF polysulphone membrane. Parallel results were obtained even with the concentrations corrected using a correction factor based on the total protein concentration at the start and at the end of dialysis as used by Lesaffer et al. (Lesaffer, 2000). Furthermore, there was no statistical difference between intradialytic start-end values, and removal efficiency for the LF and HF membranes estimated by the total area of HPLC peaks at 254 nm and 280 nm in the serum and online UV absorbance at 280 nm in the spent dialysate. This indicates that UV absorbance follows the behaviour of UV-absorbing compounds – uremic toxins – which are the origin of total UV absorbance in serum and spent dialysate.

The RR values of different identified compounds, the total area of all HPLC UV absorbance peaks on the wavelengths of 254 nm and 280 nm in the serum and the RR of online UV absorbance at 280 nm in spent dialysate are presented in Table 1 and Figure 8. Taking into account the removal efficiency, a difference can be observed in the relation of UV absorbance to small water-soluble non-protein-bound solutes and to small protein-bound solutes such as indoxyl sulphate. The small non-protein-bound solutes uric acid and urea showed a far more substantial decrease of concentration than creatinine being statistically

Optical Dialysis Adequacy Monitoring:

dialysis, uremic toxicity and kidney functionality.

**8. Acknowledgements** 

Regional Development Fund.

**9. References** 

adequacy.

**7. Conclusions** 

Small Uremic Toxins and Contribution to UV-Absorbance Studied by HPLC 157

cumulative and integrated UV absorbance curve utilised during optical dialysis dose online monitoring. The online methods are felt to be more accurate than methods based on preand post-dialysis urea concentrations, and to be better suited to clinical routine. Continuous monitoring of uremic retention solute concentrations during a dialysis session could be beneficial for the prevention of intradialytic morbidity and for the confirmation of dialysis

This chapter contributes new information about the removal of uremic retention solutes during hemodialysis and the origin of the optical dialysis adequacy monitoring signal. The relationship between characteristics of the online UV absorbance curve measured during dialysis and the identified HPLC peaks in spent dialysate was investigated. It was demonstrated that the absorbance signal reflects the contribution of several UV-absorbing compounds in spent dialysate, with the strongest influence coming from the low-molecularweight water-soluble non-protein bound compounds. Moreover, UV absorbance behaves more like small water-soluble non-protein-bound solutes than small protein-bound solutes. Monitoring the removal of compounds with different properties and elimination characteristics during various dialysis strategies adds knowledge of dialysis treatment and would be useful for future research in order to decrease complications related to dialysis quality and cardiovascular risk factors. Hopefully the online methods will add a new technique and methodology to the wide discussion about the quality and adequacy of

The authors wish to thank the nurses and technical staff who assisted us during our clinical experiments as well as the dialysis patients who kindly participated in the experiments. This study was supported by the Estonian Science Foundation (grant no. 8621), the Estonian targeted financing project SF0140027s07 and the European Union through the European

Barnett, A.L. & Veening, H. (1985) Liquid-chromatographic study of fluorescent compounds in haemodialysate solutions, *Clinical Chemistry*, Jan.31(1), pp. 127-130 Castellarnau, A., Werner, M., Günthner, R. & Jakob, M. (2010). Real-time Kt/V

Cho, D. S., Olesberg, J.T., Flanigan, M.J. & Arnold, M. (2008). Online Near-Infrared

De Smet, R., Vogeleere, P. et al. (1999). Study by means of high-performance liquid

of uremic patients, *Journal of Chromatography A*., 847(1-2), pp. 141-153

*Kidney International,* 78, November (1), pp. 920-925

*Spectroscopy*, Vol. 62, Issue 8, pp. 866-872

determination by ultraviolet absorbance in spent dialysate: technique validation,

Spectrometer to Monitor Urea Removal in Real Time During Hemodialysis, *Applied* 

chromatography of solutes that decrease theophylline protein binding in the serum

different (p < 0.05) from the RR of serum uric acid and urea. The similar removal of urea and uric acid makes it possible to use other components and methods to monitor urea reduction during a single hemodialysis session. At the same time, the RR of creatinine is statistically different (p < 0.05) from the RR of all HPLC peaks in the serum at 280 nm, but not different from that of 254 nm. The RR for online UV absorbance is lower compared to urea. Considering that RR (URR) is correlated to Kt/V (NKF-DOQI, 2006), this tendency is reported earlier, as the dialysis dose estimated by online UV absorbance was lower than Kt/V urea (Uhlin, 2003). The difference between the RR of all HPLC peaks in serum and online UV absorbance measurement (at 280 nm) in spent dialysate could be due to different chromophores in the serum and spent dialysate and because the serum was collected before and the dialysate sample 10 minutes after the start of dialysis. Moreover, the different binding of individual uremic retention solutes to serum proteins may modify percentage concentration changes of individual solutes in the course of haemodialytic treatment (Vanholder, 1992), supported by observations of decreased drug/protein interactions in uremic serum (De Smet, 1999).

The correlation analysis also provides additional insights into the removal characteristics of solutes and UV absorbance monitoring (Table 2). The RR of uric acid has the highest correlation for RR at 280 nm in both serum and spent dialysate, but not at 254 nm in serum. The explanation is the highest contribution of uric acid to UV absorbance compared to other chromophores at 280 nm (Figures 5 and 6). However, there are several other strong contributions from other compounds beside uric acid at 254 nm, and therefore the correlation is lower. The outcome in Table 2 is confirmed by comparing the millimolar extinction coefficients versus wavelengths for uric acid (Vasilevsky, 2005). A higher value of the extinction coefficient corresponds to the higher correlation for RR of uric acid at 280 nm.

The RR of urea is more related to RR at 280 nm both in serum and spent dialysate, but less so at 254 nm in serum (Fridolin, 2003). This means that relatively good correlation between the RR of UV absorbance and a particular solute may be achieved when the removal rate of a non-absorbing solute (e.g. urea) is similar to that of UV-absorbing substances during haemodialysis. This is also confirmed by very good correlation between several small molecular weight waste products and UV absorbance (Fridolin, 2002) and similar concentration changes during dialysis for several azotemic markers (e.g. urea, creatinine and uric acid) (Vanholder, 1992). The dominance of small molecular weight waste products among chromophores in serum and spent dialysate can be concluded because the number of detected HPLC peaks is not significantly different for serum filtered with a filter in cut-off 3 kDa and 70 kDa. Furthermore, it seems that the UV-absorbing solutes can be subject to similar corrections regarding distribution volume and intercompartmental equilibration rates, similar to urea, although not with exactly the same distribution and equilibration intercompartmental rates in the body as urea. This makes it possible to estimate the delivered dialysis dose in terms of Kt/V by monitoring UV absorbance in spent dialysate online (Uhlin, 2003).

The RR of creatinine demonstrates a high correlation for RR at 280 nm in both serum and spent dialysate. The reason could be similar removal of creatinine and other chromophores at 280 nm, where creatinine does not contribute significantly to UV absorbance (Figure 5).

As previous studies have shown, and as is confirmed by this work, the HPLC is a method which has its own place for the detection of uremic solutes in biological solutes. This is an effective method of studying accumulated metabolites in patients' blood and removed in dialysis. Identification of these metabolites gives us the opportunity to understand the cumulative and integrated UV absorbance curve utilised during optical dialysis dose online monitoring. The online methods are felt to be more accurate than methods based on preand post-dialysis urea concentrations, and to be better suited to clinical routine. Continuous monitoring of uremic retention solute concentrations during a dialysis session could be beneficial for the prevention of intradialytic morbidity and for the confirmation of dialysis adequacy.
