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Beads, Composite and Monolith Forms

The use of activated carbons in liver and kidney dialysis declined after early positive clinical findings. This was in part because the efficacy of the materials was lost following the introduction of coatings in order to reduce complications associated with poor biocompatibility [9-12]. Studies reported the accumulation of fine carbon particles in the lungs and significant reduction in blood platelets following haemoperfusion. The coatings reduced complications but limited accessible surface area for high molecular weight molecules. Subsequent research has revealed the significant and detrimental role played by the dysregulated build-up these high molecular weight and protein bound biotoxins in organ failure related to chronic disease and in disorders such as sepsis.

Beads

Activated carbons (ACs) derived from synthetic precursors can be prepared in different formats such as rods, pellets, beads and sheets (Fig. 9.2). However, for extracorporeal blood purification (EBP), ACs in bead form provide added benefits including consistent volume to void ratio in the packed bead column and tuneable carbon bead dimensional parameters.

Synthetic ACs prepared in (a) granulated and (b) beaded formats

Figure 9.2 Synthetic ACs prepared in (a) granulated and (b) beaded formats.

AC Beads for Protein Bound Toxin Removal

Conditions such as kidney and liver failure lead to biotoxin build up which causes further damage through sustained and unregulated inflammatory stimulus. A cycle of progressive cell and tissue damage follows which may lead to multi-organ failure and death [13,14].

Uraemic toxins accumulate in the blood stream and disrupt normal physiology during kidney failure [15]. Generally uraemic toxins are classified into three categories which are (1) free water soluble low molecular weight (<500 D) solutes such as creatinine and urea; (2) protein bound solutes and (3) middle to large molecules (>500 D) [16]. The levels of free water soluble low molecular weight uraemic toxins such as creatinine and urea have not only been used as biomarkers for uraemic syndrome, but are also used as important indicators of the effectiveness of renal replacement therapy. However, increasing clinical evidence suggests that the toxicity of these two compounds is relatively low compared to albumin bound and middle to large molecular weight toxins [17, 18]. Two examples of albumin bound uraemic toxins are p-cresyl sulphate (p-CS) and indoxyl sulphate (IS). They are poorly removed by current renal replacement therapies and are linked to cardiovascular complications common in patients with chronic kidney disease [19, 20]. Studies have suggested the inclusion of serum p-CS and IS levels as predictors of CKD progression and related clinical complications [18,21].

Similarly biotoxins in liver failure can also be categorised into three groups:

  • (1) small molecular weight water soluble toxins such as ammonia, lactate and urea;
  • (2) middle and high molecular weight water soluble toxins such as amino acids, peptides and interleukins;
  • (3) albumin-bound toxins such as bilirubin, bile acids aromatic amino acids, indoles.

Bilirubin is a classic liver failure bio-marker and notoriously difficult to remove due to its high albumin binding affinity. The majority of key hepatic toxins are albumin bound meaning that conventional dialysis alone is not sufficient for the treatment of liver failure. Albumin bound liver toxins have been targeted for removal using EBP systems such as single pass albumin dialysis (SPAD), Molecular Adsorbent Recirculating System (MARS®, Gambro) [22, 23] and Prometheus™ (Fresenius Medical Care) [24, 25].

EPB systems currently in use for kidney failure remove smaller toxins associated with CKD such as urea and creatinine. Techniques such as haemodialysis and peritoneal dialysis are lifesaving treatments for patients with end stage CKD. However, the removal of high molecular weight toxins and a wide selection of protein bound toxins remains a major challenge in the optimisation of these systems [12]. Although the reduction of some middle molecular weight molecules and albumin bound toxins in a patient's blood circulation can be achieved through albumin dialysis (MARS system) or albumin containing plasma filtration (Prometheus system), the relatively low removal of albumin bound toxins and the high cost associated remain some of the limiting factors in the use of these techniques. Evidence suggests that some of these albumin bound toxins have the most damaging effects but are poorly removed by current devices [13]. Effective adsorbents that are capable of removing such biotoxins may provide additional treatment tools to improve the management of end stage kidney and liver failure. A selection of marker albumin bound biotoxins used in adsorption studies indicating their size and protein binding characteristics is shown in Table 9.1.

Table 9.1 A selection of marker albumin bound biotoxins used in adsorption studies indicating their size and protein binding characteristics

Biotoixns

Associated

conditions

Molecular weight (g/inole)

Albumin binding constant (M_1)

Tryptophan

Liver failure

204.23

1.0 x Ю4

Cholic acid

Liver failure

408.57

0.33 X Ю4

Bilirubin

Liver failure

584.66

9.5 x Ю7

p-cresyl sulphate

Kidney failure

188.20

1.0 x 10s

Indoxyl sulphate

Kidney failure

213.21

0.98 x 10s

Although the adsorptive properties of AC are well studied, the mechanism of AC adsorption of albumin bound molecules remains largely unclear. Studies suggested that the efficient removal of albumin bound toxins by haemosorbent granulated deliganding (HSGD) ACs could be due to the deliganding effects of AC, leading to the purification of albumin from high affinity toxins such as unconjugated bilirubin [26]. Furthermore, the HSGD carbon also demonstrated efficient removal of relatively low affinity uraemic toxins including 3-carboxy-4-methyl-5-propyl-2-furanpropionate (albumin association constant 130 * 10s M"1), indoxyl sulphate (albumin association constant 16 x Ю5 M_1) and hippuric acid

(albumin association constant 0.1 x 10s M"1) [27]. Mesoporosity of the HSGD ACs was reported to assist the AC deliganding and adsorption of albumin bound toxins [28].

Similar to HSGD carbons, NovaCarb S (Mast Carbon International Ltd, UK) is also prepared by the pyrolysis of synthetic resins and possesses a large surface area. Furthermore, a study conducted by Tripisciano, et al. suggested the removal of albumin bound liver toxins including bilirubin, cholic acid, tryptophan and phenol was dependent on the degree of burn off during the AC pyrolysis process [6]. Further studies have provided a more comprehensive understanding regarding the dependence of albumin bound toxin adsorption efficiency on the pore size distribution of NovaCarb S ACs.

To investigate the relationship between AC pore size and removal of marker albumin bound toxins, a section of synthetic AC beads with similar particle sizes (250 to 500 pm in diameter), large specific surface are (>1200 m2/g), abundant micropores and increasing pore size in the meso- to macropore range (Table 9.2) were used.

Table 9.2 Characteristics of ACs used in liver and kidney toxin removal studies. D is pore sizes (diameter) determined by mercury porosimetry; D is the AC bulk density; Fmjcro is the volume of pores less than 2 nm diameter; SBET is the BET specific surface area determined by nitrogen adsorption. (Data obtained from the manufacturer)

d (nm)

D

(g/ml)

V

r micro

(cin3/g)

V

r micro

(cm3/ml)

Sbet

(m2/g)

  • •^bet
  • (m2/ml)

Al

<2 nm

0.56

0.69

0.39

1204

674

A2

30

0.38

1.30

0.49

1559

592

A3

70

0.27

1.75

0.47

1493

403

A4

80

0.21

1.80

0.38

1548

325

Using a series of AC beads with increasing meso- to macropore size, the study demonstrated that all of the selected synthetic AC beads can remove small molecular weight albumin bound toxin markers with lower binding affinity evidenced using marker molecules IS (Fig. 9.3A) and p-CS (Fig. 9.3B).

Amount of (A) indoxyl sulphate and

Figure 9.3 Amount of (A) indoxyl sulphate and (B) p-cresyl sulphate remaining in spiked plasma after contacting AC1-4. The removal of p-cresyl sulphate by 0.5 g microporous carbon (Al), mesoporous carbon (A2) and macroporous carbon (A3 and A4) from 10 ml 250 pM (0.25 pmol/ml) p- cresyl sulphate spiked plasma was observed at 4 time points over 60 min incubation period. (Mean n = 4, ± SEM). *** represents p < 0.001 in Bonferroni post-test of two-way-ANOVA statistical analysis.

The batch adsorption studies by AC adsorption of albumin bound hepatic and uraemic toxins revealed the efficient removal of toxins such as tryptophan, phenol, cholic acid, indoxyl sulphate and p-cresyl sulphate by all the tested ACs. In addition, to their high adsorption capacity particularly for the phenolic toxins, ACs with mean pore diameter of 80 nm showed an ability to remove bilirubin from the spiked plasma samples (Fig. 9.4). A4 carbon with a mean pore diameter of 80 nm and 1550 m2/g specific surface area demonstrated the most efficient liver toxin removal.

Adsorption of albumin bound toxins by ACs could involve complex mechanisms such as binding competition among toxins and possible deliganding of toxins from albumin by the AC surfaces. However, little work has been done to improve understanding of the mechanisms involved. The results support the use of ACs to remove these marker uraemic and liver toxins and other structurally related biotoxins given the non-specific nature of AC binding.

Amount of (A) albumin and (B) bilirubin removed from plasma samples

Figure 9.4 Amount of (A) albumin and (B) bilirubin removed from plasma samples. Samples were collected after 0.6 ml of AC beads (A-l, A-2, A-3 and A-4) were incubated with 5.4 ml of bilirubin (0.3 pmol/ml) spiked fresh frozen human plasma for 5, 15, 30 and 60 min, and analysed using Roche/Hitachi 902 Chemistry Analyzer (Mean n = 5, ± SEM). p < 0.001% Bonferroni post-test of two-way-ANOVA statistical analysis.

 
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