New Approaches to the Removal of Protein-Bound Toxins from Blood Plasma of Uremic Patients

Figures & data

Table 1. Binding capacity of albumin isolated from blood plasma of uremic patients

Medicinal preparation	Binding center for pharmaceuticals by Sudlow G et al. [[2]]	Binding capacity of albumin	Reference
Digitoxin	III	Decreased	[[3]]
Kass = 4.3 × 10^4 M^−1; n = 1
Varfarin			[[4]]
Kass1 = 2.3 × 10^5 M^−1 ; n1 = 1	I	Decreased
Kass2 = 5.9 × 10^3 M^−1; n2 = 4	III
Diazepam	II	Decreased	[[5]]
Kass = 4.9 × 10^5 M^−1; n = 1
Salicylic acid	I	Decreased	[[6]]
Kass1 = 7.1 × 10^4 M^−1; n1 = 1	III
Kass2 = 3.3 × 10^3 M^−1; n2 = 4
Pnenylbutazone	I	Decreased	[[7]]
Kass1 = 1.0 × 10^5 M^−1; n1 = 1
CKss2 = 4.0 × 10^4 M^−1; n2 = 2
Indomethacin	III	Decreased	[[8]]
Kass1 = 1.0 × 10^5 M^−1; n1 = 1
Kass2 = 4.0 × 10^4 M^−1; n2 = 2

K_ass-constant of association with albumin molecule; n-number of binding sites.

Table 2. Protein-bound uremic toxins

2-methoxyresorcinol	p-cresol
3-deoxyglucozone	Pentosidine
CMPF	Phenol
Fructoselysine	p-OH-hippuric acid
Glyoxal	Retinol-binding protein
Hippuric acid	Spermidine
Homocysteine	Spermine
Indoxyl sulphate	Leptin
Kynurenine	Melatonin
Kynurenic acid	N^ε-(carboxymethyl)lysine

Figure 1 Melting thermograms of defatted HSA before (a) and after (b) loading with protein-bound uremic toxins. Dotted lines describe the results of this curve mathematical deconvolution onto elementary peaks.

Figure 2 Average melting thermograms of defatted HSA and CMPF-HSA complex (M:M = 2:1).

Table 3. Parameters of complex-formation for HSA with the 3 typical protein-bound uremic toxins

Uremic toxin	n1	Kass = (10^5 × M^−1)	n2	Kass = (10^3 × M^−1)
	1	130,5	2	33,4
	1	16,1	3	8,3
	1	0,1	7	0,3

Table 4. Enthalpy of complex-forming abilities (kJ/M) of albumin preparations with marker ligands with respective affinity to binding centers of protein

Sample	Sodium octanoate (K1 = 5.5 × 10^5 M^−1, n1 = 1	Salicylic acid (K1 = 1.99 × 10^5 M^−1, n1 = 1	Phenol red (K1 = 0.28 × 10^5 M^−1, n1 = 1
Defatted albumin	25.5 ± 0.50	31.5 ± 0.50	11.20 ± 0.30
Defatted albumin + HA (Kass HA = 0.5 × 10^5 M^−1, n1 = 1)	23.2 ± 0.60	26.8 ± 1.40	6.50 ± 0.40
Defatted albumin + IS (Kass IS = 16.0 × 10^5 M^−1, n1 = 1)	0.06 ± 0.03	20.3 ± 0.60	3.40 ± 0.10
Defatted albumin + CMPF (Kass CMPF = 4.8 × 10^6 M^−1, n1 = 1)	25.4 ± 0.80	32.5 ± 1.10	12.20 ± 0.50

Figure 3 Melting thermograms of albumin fraction isolated from blood plasma of uremic patients undergoing chronic alimentary hemodyalysis for 6 months–20 years.

Table 5. Enthalpy of complex-formation of marker ligands (ΔHc, kJ/Mol) with albumin of healthy donors and uremic patients undergoing hemodialysis of different duration

	Ligand
Sample	Sodium octanoate	Salicylic acid	Phenol red	Bromsulphalein	SDS
HSA of healthy donors	19.1 ± 0.3	27.4 ± 1.0	6.1 plusmn; 0.2	47.3 ± 0.8	66.4 ± 1.0
Uremic HSA
Less than 1 year on HD	9.0 ± 0.4^a	17.7 ± 0.9^a	3.8 ± 0.1^a	30.7 ± 1.1^a	43.0 ± 1.0^a
1–5 years on HD	5.1 ± 0.2^a,^b	15.0 ± 0.5^a,^b	2.2 ± 0.1^a,^b	2.2 ± 1.2^a	45.2 ± 0.5^a
5–10 years on HD	10.8 ± 0.4^a,^b	19.0 ± 0.3^a,^b	2.9 ± 0.1^a,^b	2.8 ± 0.9^a	45.7 ± 0.8^a
15–20 years on HD	15.4 ± 0.4^a,^b	24.9 ± 0.7^a,^b	3.9 ± 0.2^a,^b	37.7 ± 1.2^a,^b	47.5 ± 0.7^a

^ap < 0.001 compared to HSA of healthy donors.

^bp < 0.001 compared to uremic HSA from the previous group.

Table 6. Enthalpy of complex formation (kJ/M) of albumin preparations with marker ligands for respective affine binding centers of protein

Sample/ligand	Sodium octanoate	Salicylic acid	Phenol red	Bromsulphalein	SDS
HSA of healthy donor	18.95	27.63	6.47	56.25	65.57
Uremic HSA before HD	10.38	18.44	3.03	40.74	44.77
Uremic HSA after HD	5.08	12.54	2.97	39.18	40.03
Uremic HSA upon PD	10.3	19.17	2.49	40.74	62.71

Figure 4 Demonstration of inefficacy of exhaustive dialysis and contact with SCN carbons of blood plasma of uremic patients for the removal of protein-bound toxins obtained by the methods of differential (a, b, c) and flow microcalorimetry (d).

Table 7. Influence of adsorptive purification of uremic plasma at different pH on the value of enthalpy (kJ/M) of complex formation of uremic HSA with marker ligands

			PH
Ligand	Patient	Original uremic HSA	3	4	5	6	7	8	9	HSA of healthy donors
Sodium octanoate	1	0.30	20.68	15.12	5.15	1.47	0.56	0.23	1.69	18.55
	2	0.20	17.04	15.96	8.40	3.19	0.05	1.81	0.25
Bromsulphalein	1	35.03	41.21	37.39	43.14	36.99	36.27	38.86	38.70	42.28
	2	36.62	39.40	38.80	36.37	30.15	37.29	38.70	38.91
Salicylic acid	1	11.35	30.01	25.71	19.84	17.87	14.72	14.30	15.54	24.73
	2	12.46	27.50	26.99	21.60	19.84	17.61	19.50	17.58
Phenol red	1	3.95	6.24	4.99	4.32	3.57	3.18	4.01	5.08	5.01
	2	3.01	7.02	4.70	3.90	3.01	4.52	4.01	3.82
Sodium dodecyl sulphate	1	32.80	67.37	52.18	38.48	36.94	29.95	50.22	39.47	72.81
	2	31.03	57.84	57.99	47.26	41.17	30.49	39.61	34.44

Figure 5 Melting thermograms of HSA isolated from uremic plasma before (a) and after adsorptive purification at pH = 7.2 and 3.0 (b and d), and after dilution with acetate buffer (pH = 5.08) with the next purification on carbonic adsorbent (c).

Table 8. Enthalpy of complex formation (kJ/M) of marker ligands with preparations of HSA isolated from uremic plasma treated by purification on the adsorbent at different pH values and acetate buffer dilution (ACB)

	Ligand
Sample	Sodium octanoate	Salicylic acid	Phenol red	Bromsulphalein	SDS
HSA of healthy donors	18.95	27.63	6.47	56.25	65.57
Uremic HAS
Original	9.63	20.28	4.21	48.05	54.18
pH = 7.4	16.99	26.49	6.15	51.59	63.23
pH = 5.08 ACB dilution	21.15	30.85	5.87	55.35	68.87
pH = 3.0	18.96	28.15	5.95	56.01	66.62

Figure 6 Principal scheme for the purification of blood plasma of uremic patients with the use of acetate buffer.

Table 9. Transformation of conventional phenolformaldehyde carbon (sample 1) into deliganding form (sample 2)

Bulk weight, g/cm^3	Adsorption of creatinin (M.w. = 113.1), mg/g	Adsorption of vitamin B12 (M.w. = 71355), mg/g	Adsorption of bilirubin (M.w. = 584), from 3% HSA solution, mg/g
0.33, sample 1	52.5	162.5	0.56
0.168, sample 2	52.7	191.2	4.71

Table 10. Correlation between the degree of fractality of carbonic adsorbent (α) and adsorption of nonconjugated bilirubin (mg/g) from albumin-containing solution

Coefficient α	Type of structure	Adsorption of bilirubin (mg/g)
−3.51	Fractal surface	0.82
−2.99	Mass-fractal	5.20
−2.80	Mass-fractal	13.40
−2.39	Mass-fractal	98.70

Figure 7 Melting thermograms of CMPF-HSA complex before (1) and after (2) the contact with deliganding HSGD haemosorbent (a) and decrease of concentration of CMPF, indoxyl sulphate (IS) and hippuric acid (HA) after sorptional purification on HSGD carbons in microcolumn experiments (b).

Figure 8 Melting curves and complex forming ability of HSA obtained from blood plasma of healthy donors and non-diabetic uremic patients preliminarily treated on HSGD haemosorbents.

Figure 9 Principal scheme for the removal of protein-bound uremic toxins from blood of patients with renal insufficiency (from patient – pump 1 300 ml/min – pump 2, 30 ml/min – three-substituted citrate – deliganding adsorbent – to patient – dialysator).

Table 11. HSGD carbons in the therapy of heavy forms of poisoning of children with death cap

Type of extracorporeal therapy	Number of patients	Number of lethal outcomes	Lethality (%)
Conventional SCN haemocarboperfusion	29	17	58.6
Haemocarboperfusion onto HSGD adsorbents	19	2	10.5

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