Detection and adequacy evaluation of erythrocyte glutathione transferase on levels of circulating toxins in hemodialysis patients

Abstract

To explore detection and adequacy evaluation of erythrocyte glutathione S transferase (GST) on levels of circulating toxins in hemodialysis patients in Qinhuangdao region in China, this study divided 84 cases of long-term, end-stage hemodialysis patients into 2 groups: one group of 33 cases of adequate hemodialysis (spKt/V ≥ 1.3) and another group of 51 cases of inadequate hemodialysis (spKt/V < 1.3), according to the urea index value of the unit chamber model (spKt/V). Another 50 cases of subjects found healthy by a physical examination were taken as the control group, and the differences in the related clinical and biochemical indexes of the 3 groups were compared and analyzed. The levels of GST, creatinine, high sensitivity C-reactive protein (hs-CRP), transferrin saturation (TSAT), parathyroid hormone (PTH), interleukin-2,6,8 (IL-2,6,8) and tumor necrosis factor-a (TNF-a) in the hemodialysis group were significantly higher than those in the control group (P < 0.05), and GST, IL-2, 6, 8, and TNF-a levels in the inadequate hemodialysis group were significantly higher than in the adequate hemodialysis group (P < 0.05). Pearson's relevant analysis showed that the levels of GST and spKt/V, IL-2, IL-6, IL-8, and TNF-a have a positive correlation (P < 0.05), and they have no correlation with levels of creatinine, hs-CRP, TSAT, and PHT (P > 0.05). There were 23 patients with levels of spKt/V ≥ 1.3 after adjusting the dialysis solution for 51 cases of inadequate hemodialysis patients, and the GST level after the adjustment was significantly lower than that before the adjustment, but still higher than that in the adequate dialysis group. This concludes that the maintenance of hemodialysis in patients has certain relevance on spKt/V and associated inflammatory factors. Through the study, it can be determined that GST can effectively respond to adequate hemodialysis, which has a guiding significance on adjusting the blood dialysis solution in clinical practice.

Keywords:

• Dialysis adequacy
• erythrocyte glutathione transferase
• hemodialysis
• inflammatory factor

Introduction

Hemodialysis is one of the most effective means for continuing the life of end-stage renal disease patients. Sufficient dialysis can protect against protein loss in the patient, and plays an important role in enhancing the body’s nutritional status and resistance (Wu et al. 2011). Erythrocyte glutathione S transferase (GST) can be involved in various physiological reactions and has been known and recognized in a variety of diseases, especially with the correlation of tumor, but evaluation research is relatively less regarding GST as an index of adequate hemodialysis (Xie 2012). Recently, foreign scholars Dessi et al. (2012) have confirmed that GST and inflammation in end-stage renal disease patients have certain relevance. In addition, Bonifazi et al. (2014) conducted a study on the relationship between GST and kidney damage, and pointed out that the GST level in patients with kidney disorder is significantly higher than that in normal people, therefore both have a close relationship. Seabra and Perianayagam (2011), in their study, showed that due to inadequate hemodialysis in patients, complete elimination of toxins from the body is difficult, which leads to the accumulation of a large number of toxins in the body, and stimulates the generation of GST in great quantities, raising the level of serum GST. The above research put forward a corresponding basis for GST as an adequate index for hemodialysis evaluation.

GST is an index of the levels of circulating toxins in hemodialysis patients. The patient's GST level is relatively stable in the short-term. Therefore, the expression of the index in the short term is insufficient and limited, but the expression in a longer period is more significant. In a period of 2 months, the index showed an evaluatory function similar to that of the level of glycosylated hemoglobin as an index for blood glucose levels over a long term, which is one of the important performances of GST as a biomarker for uremic toxicity and adequacy of dialysis (Liu and Zhan 2014).

Based on the above foundation, this study detected and analyzed the various indexes of the groups with adequate and inadequate hemodialysis, and the indexes of the healthy group, to explore detection and adequacy evaluation of GST on levels of circulating toxins in hemodialysis patients.

Materials and methods

General data

Since the selection areas were limited, this experiment selected 84 cases of patients who received maintenance hemodialysis between May 2011 ∼ May 2013 from a hospital as the research subjects, of which 52 cases were of men and 32 cases were of women, ranging in age from 38 to 78 years (mean 52.3 ± 1.4 years), with anaphylactic purpura nephritis in 11 cases and lupus nephritis in 13 cases. The patients were divided into one group of 33 cases with adequate hemodialysis (spKt/V value ≥ 1.3) and another group of 51cases of inadequate hemodialysis (spKt/V value < 1.3), according to the spKt/V value. Another 50 cases of healthy controls were taken as the control group, of which 25 cases were of males, and 25 cases were of females, ranging in age from 18 to 75 years (mean 49.26 ± 2.1 years).

The inclusion criteria of patients on maintenance hemodialysis: 1) Hemodialysis time is over 3 months; 2) the patient is in stable condition; 3) signed informed consent. Exclusion criteria for patients: acute cardiovascular complications, recent infections or bleeding, application of immunosuppressant for nearly 3 months, malignant tumor, thyroid dysfunction, and patients with acute or chronic liver disease.

Test method

After centrifugal processing of 3 ml of venous blood collected from the subjects early in morning on an empty stomach, the ELISA enzyme-linked immunoassay assay was used to determine the levels of IL - 2, IL - 6, IL - 8 and TNF -a (Shanghai Biological Co., LTD) respectively; immunoturbidimetry was performed to determine hs-CRP (Suzhou Keming Biological Technology Co., LTD); ultraviolet spectrophotometry was performed to determine GST (B&G USA); a Hitachi 7600-020 automatic biochemical analyzer was used to determine creatinine, hemoglobin, TSAT, and PTH.

Hemodialysis adequacy evaluation index

Including a patient's clinical status, weight loss status, Kt/V value, single room Kt/V value (spKt/V) = -Ln(R-0.008t) + (4-3.5R) UF/W. A spKt/V value ≥ 1.3 signifies adequacy of hemodialysis.

Statistical testing

Data analysis was performed by SPSS17.0, one-way variance and LSD test. Enumeration data and group enumeration were expressed by rate and x², respectively. Pearson’s correlation analysis was done and all results were statistically significant if P ≤ 0.05.

Experimental result

Analysis of the clinical data and biochemical indicators of each group

The results of detecting and analyzing the clinical data and biochemical indicators of the adequate hemodialysis group, inadequate hemodialysis group, and the control group with healthy results in the physical examination showed that the levels of creatinine, hs-CRP, PTH, TSAT, IL - 2, IL - 6, IL - 8, TNF - a, and GST in the inadequate hemodialysis group were significantly higher than those in the adequate hemodialysis group and the healthy control group, especially in the healthy control group; a comparison of the hemoglobin level between the 3 groups showed that the level in the inadequate hemodialysis group was significantly lower than the levels in the other 2 groups; the specific numerical value is as shown in Table 1.

Table 1. Clinical data of each group and biochemical indicator analysis ( + s).

Index	Adequate hemodialysis group (n = 33)	Inadequate hemodialysis group (27/24)	Control group (n = 50)	X^2/F	P value
Gender (male/female)	19/14	27/24	26/24	0.396	> 0.05
Age (g)	52.96 ± 1.29	52.34 ± 1.39	49.26 ± 2.1	0.985	> 0.05
Creatinine (mg/dL)	11.26 ± 2.36a	12.45 ± 2.85a	0.98 ± 0.46	4.985	< 0.05
Hemoglobin (g/Dl)	11.98 ± 1.62a	10.61 ± 1.74a	14.98 ± 2.62	5.885	< 0.05
Hs-CRP (mg/L)	7.02 ± 1.63a	9.26 ± 2.32a	0.38 ± 0.06	12.984	< 0.05
PTH (pg/mL)	192.32 ± 16.87a	210.32 ± 20.69a	0.19 ± 0.06	38.692	< 0.05
TSAT (%)	18.69 ± 3.45a	20.12 ± 7.32a	0.69 ± 0.06	11.002	< 0.05
IL-2 (mmol/L)	24.69 ± 10.39a	35.36 ± 11.36ab	7.19 ± 2.03	10.362	< 0.05
IL-6 (mmol/L)	7.96 ± 1.06a	9.36 ± 2.58b	2.03 ± 1.05	9.742	< 0.05
IL-8 (mmol/L)	12.21 ± 2.89a	18.63 ± 3.05a	3.29 ± 1.03	9.326	< 0.05
TNF-a (mmol/L)	21.36 ± 8.25a	27.33 ± 4.98a	4.26 ± 1.08	8.669	< 0.05
GST (U/L)	20.32 ± 3.78a	38.19 ± 4.52ab	10.32 ± 1.26	6.785	< 0.05

Note: Compared with the control group, ^aP < 0.05.

Compared with the adequate hemodialysis group, ^bP < 0.05.

Correlation analysis of GST and hemodialysis degree and the inflammation factor

The correlation analysis of GST and hemodialysis degree and the inflammation factor by Pearson's single factor showed that the P value of spKt/V,IL-2, IL-6, IL-8 and TNF-a was lower than 0.05, which had positive correlation, but there was no correlation with creatinine, hs-CRP, and TSAT(P > 0.05). The specific numerical value is as shown in Table 2.

Table 2. Correlation analysis of GST, degree of hemodialysis, and the inflammation factor.

	spKt/V	IL-2	IL-6	IL-8	TNF-a	Creatinine	Hs-CRP	TSAT	PTH
r	0.478	0.426	0.468	0.433	0.418	0.112	0.164	0.137	0.145
P	0.000	0.004	0.002	0.001	0.005	0.078	0.092	0.084	0.069

Adjusted hemodialysis index analysis

The results of detection and analysis of SpKt and GST before and after adjustment in the hemodialysis group and inadequate hemodialysis group showed that before and after the adjustment in the adequate hemodialysis group of 33 cases, the patients’ SpKt values were 1.3 or higher, but the GST value decreased slightly after the adjustment. After the adjustment in the inadequate hemodialysis group of 51 cases, the patients’ SpKt values were down to < 1.3 in 28 cases, SpKt ≥ 1.3 instead of 0 in patients up to 23 cases, and the numerical value of GST presented a downward trend. Comparing the 2 groups of patients, we found that regardless of whether the evaluation was done before or after the adjustment, the GST value of the inadequate hemodialysis group was higher than that of the adequate hemodialysis group. The specific numerical value is as shown in Table 3.

Table 3. Analysis of hemodialysis index before and after adjustment.

		Before adjustment			After adjustment
Group	Cases	SpKt < 1.3	SpKt ≥ 1.3	GST	SpKt < 1.3	SpKt ≥ 1.3	GST
Adequate hemodialysis group	33	0	33	20.32 ± 3.78	0	33	18.62 ± 3.74
Inadequate hemodialysis group	51	51	0	38.19 ± 4.52	28	23	30.12 ± 5.32ab

Note: Compared with the control group, ^aP < 0.05.

Compared with the adequate hemodialysis group, ^bP < 0.05.

Discussion

Maintenance hemodialysis becomes integrated therapeutic treatment for patients with end-stage renal failure and renal replacement therapy; now a large number of studies have shown that the prognostic effect for patients with maintenance hemodialysis is closely related to the degree of the dialysis, and adequate dialysis has important significance in improving the quality of survival in patients with end-stage renal disease and in prolonging the survival period (Wang 2013). The Kt/V value is currently one of the important indicators for assessing the adequacy of a patient's hemodialysis. However, because the Kt/V value can only be used to reflect the clearance of small molecules and cannot effectively reflect the removal of macromolecular material, there are some shortcomings in accuracy and sensitivity, therefore, in the clinical assessment of hemodialysis adequacy, there should be comprehensive indicators in order to improve the accuracy (Li et al. 2012, Huang et al. 2012). GST is an enzyme involved in the second phase reaction of biological conversion in the liver, which can combine with a variety of halogenated compounds and epoxy compounds; its main effect is to catalyze and restore glutathione thiol and combine hydrophobic material, to enable the conversion of electrophilic compounds into hydrophilic compounds to be excreted through the urine or bile, and therefore plays the role of detoxification (Liu 2011, Chen et al. 2013). Besides GST, regulatory substances such as hs-CR, IL-2, 6, 8 and TNF-a, etc. are taken as indexes for comprehensive exploration, thus enhancing accuracy.

Urinary toxins, micro inflammation, and nutritional status are the main problems that lead to a variety of complications in dialysis patients and threaten the long-term survival rate and survival quality (Xie 2012). Dialysis can clear the water retention generated during the dialysis interval (reaching dry weight), remove the toxins generated by decomposition and metabolism of the proteins, and correct electrolyte and acid-base balance disorders, which enable people to feel comfortable to the point of psychological rehabilitation and return to society (Liu 2011). This study showed that the GST level in the hemodialysis group was higher than that in the control group, which to some extent indicated that GST can be the basis of adjustments in the process of hemodialysis. Through further analysis of the adequate dialysis group and the inadequate dialysis group, we found that the GST level in the inadequate dialysis group before and after the adjustment is also higher than that of the adequate dialysis group, and levels of IL - 2, IL - 6, IL - 8 and TNF - a are significantly higher than those in the adequate hemodialysis group, which may result from the loss of large amounts of protein seen in patients with inadequate hemodialysis, thus leading to the appearance of the symptoms of chronic malnutrition and thereby weakening immunity and causing a rise in the patients’ inflammation levels. In addition, the study showed that the levels of GST and IL - 2, IL - 6, IL - 8, and TNF - a have positive correlation, which suggested that GST can be used as a predictor of inflammatory level in patients with end-stage renal disease. The essence of micro inflammation in immune inflammation is the activation of the inflammation state that the body activates through the mononuclear macrophage system to cause TNF-a proinflammatory factor in the stimulation of a variety of chemicals, endotoxins, immune complex, and alexin (Xie 2012). Studies have shown that inadequate dialysis, microinflammation of the blood dialysis medium, and small molecular toxins may be one of the key factors of malnutrition in patients (Lan et al. 2011); high flux hemodialysis can better clear the leptin and other toxic molecules in the plasma, to improve the nutritional status of dialysis patients, which concurs with the research results.

This paper argues that GST complements spKt/V or even replaces it as a new tool of evaluation of the effect of dialysis: ST is highly expressed as an endogenous biological factor when the hematotoxic level increases; ' In contrast to the spKt/V parameters, the red blood cell is a nuclear-free cell, therefore its inner enzyme content is obtained before maturity or in the process of maturity. The content of mature cells is constant in the process of survival, which can reflect the related expression of the lifetime of the circulating red blood cells in 1 ∼ 2 months (Basile and Lomonte 2012); 'GST has no correlation with other parameters (spKt/V, albumin, and urea, before and after dialysis), thus it will not interfere with the result of the experiment.

GST can achieve cell protection through the combination of a variety of toxin and active-response endotoxin levels in the body (Liu et al. 2014).The above research showed that the GST level and the spKt/V of patients with maintenance hemodialysis and associated inflammation factors have certain relevance. It can effectively reflect the adequacy of blood dialysis by detecting GST, and can also reflect the level of circulating toxins well, as a biomarker of hematotoxicity.

Acknowledgement

This research was sponsored by the People's Republic of China: BSY13C002.

Disclosure statement

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.