Chapter 1 Hemoglobin Release Test: Starch-Agarose Mixed Gel Electrophoresis

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1 Chapter 1 Hemoglobin Release Test: Starch-Agarose Mixed Gel Electrophoresis Yan Su 1,2, Lijun Gao 2 and Wenbin Qin 2 * 1 Department of Biochemistry and Molecular Biology, Baotou Medical College, China 2 Laboratory of Hemoglobin, Baotou Medical College, China * Corresponding Author: Wenbin Qin, Laboratory of Hemoglobin, Baotou Medical College, 31#Jianshe Road, Donghe District, Baotou, Inner Mongolia, , China, Tel: or ; Fax: ; qinwenbinbt@sohu.com First Published October 16, 2017 Copyright: 2017 Yan Su, Lijun Gao and Wenbin Qin. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source. 2

2 Abstract Hemoglobin release test (HRT) is performed by electrophoresing erythrocytes directly on the starch-agarose mixed gel. HRT can be divided into initial release HRT and re-release HRT according to whether the power outage is simulated, or into isotonic HRT and hypotonic HRT according to whether the sample is needed to be diluted with H 2 O. In this study, HRT was used to detect the rereleased Hb from different diseases including thalassemia, hereditary spherocytosis (HS), carcinoma, uremia and general surgery patients. The results showed that different disease all can change the amount of re-released Hb from erythrocytes. To elucidate the mechanism, the effects of H 2 O 2, glutaraldehyde and plasma components on HRT were observed in vitro. Experimental results showed that the amount of re-released Hb depends on the interaction between erythrocytes membrane and Hb, abnormalities of membrane protein, Hb and plasma environment can lead to the change of re-released Hb during HRT. So, the detection of re-released Hb has important clinical meaning, and HRT might become a useful method for diagnosis of some diseases. Introduction Gel electrophoresis is a method for separation of macromolecules (DNA, RNA and proteins), based on their size and charge. Starch gel is one of a wide variety of nontoxic supporting media that can be used for horizontal 3

3 zone electrophoresis [1]. The gels are slightly more opaque than acrylamide or agarose. Non-denatured proteins can be separated according to charge and size. They are visualized by using Napthal Black or Amido Black staining. Typical starch gel concentrations are 5% to 10%. Agarose gels do not have a uniform pore size, but are optimal for electrophoresis of proteins that are larger than 200 kda. Starch-agarose mixed gel is not commonly used in protein separation, some researchers use it for isoenzymes and lipoproteins separation [2-3]. In our lab, starch gel was first used to isolate hemoglobin (Hb), and then proper amount of agarose was added into the starch gel to increase the resolution. Now, starch-agarose mixed gel becomes the routine method for Hb research in our laboratory. Establishment of Hemoglobin Release Test (HRT) HRT is performed by electrophoresing erythrocytes directly on the starch-agarose mixed gel, on which Hb can be separated into HbA and HbA 2 [4]. In 2007, a sudden power outage was encountered during erythrocytes electrophoresis, however, the experiment was not abandoned and electrophoresis was continued after the power was restored. To our surprise, another new Hb band was found to be released from the origin, and this phenomenon was called Hb re-release [5]. 4

4 Classification of HRT HRT is divided into initial release HRT and re-release HRT, which include pointing release electrophoresis, multiple band or ladder band release electrophoresis and bidirectional ladder band release electrophoresis. In addition, HRT can also be divided into isotonic HRT and hypotonic HRT according to whether the sample is needed to be diluted with H 2 O. Initial Release and Re-Release HRT Initial Release HRT The power supply is continuous, and Hb within erythrocytes would be released only once. During initial release, the difference in the mobility of HbA 2 was found between erythrocytes and hemolysate samples, which was called HbA 2 phenomenon [6]. Re-release HRT Single-Band Re-Release HRT The power outage is simulated only once, and single Hb band appears between HbA and origin. Multiple-Band Re-Release HRT or Ladder- Band Re-Release HRT The power outage is simulated more than once, and multiple Hb bands appear between HbA and origin. 5

5 One-Direction and Double-Direction HRT One-Direction HRT The HRT electrophoresis is run only in one direction. Double-Direction HRT Two vertical-direction HRT electrophoresis are performed. The first directional electrophoresis is similar to that of ladder-band re-release HRT. After the first-direction electrophoresis, the direction of the electric field is altered vertically and another run of HRT electrophoresis is performed. Isotonic and Hypotonic HRT The electrophoresis method was the same as onedirection ERT, but the erythrocyte suspension and whole blood needed to be diluted with H 2 O in the proportion from 10: 0 to 1: 9 (named as tube 1 to 10 respectively), and then kept them at room temperature or 37ºC for 1 hour. Then one-direction HRT was performed as described above. Material and Method Preparation of Erythrocytes Suspension The anti-coagulated blood was centrifuged at 3000 rpm for 5 minutes and the upper plasma was aspirated. 200 µl of the lower erythrocytes was transferred to 1.5 ml Ep tube and then 1mL of physiological saline was added. 6

6 After gently mixing, the sample was centrifuged at 3000 rpm for 4 minutes and the supernatant was aspirated. This washing procedure was repeated for 4-5 times until the supernatant becomes colorless and transparent. Following aspirating the supernatant, the erythrocytes were diluted with saline at the ratio of 1:1 to prepare erythrocytes suspension. Preparation of Hemolysate 100 µl CCl 4 was added to 400 µlwell prepared erythrocytes suspension. The sample was centrifuged at rpm for 10 minutes after being vortexed for at least 5 minutes, and then the upper red hemolysate was transfered carefully to a new 1.5 ml Ep tube and stored at 4ºC for later use. Preparation of the Starch-Agarose Mixed Gel 1g of starch and 0.15g of agroase was dissolved with 50 ml of 1 TEB. The gel solution was heated on the electric stove repeatedly until the starch and agroase were resolved completely. Then the gel solution was poured onto a 17 cm 10 cm horizontal glass plate after being cooled down to about 60 C. Starch-Agarose Electrophoresis 8µL of erythrocytes suspension was added on a 0.1 cm 1 cm 3MM filter paper, and then was inserted into the gel which was about 2.5 cm away from one side of the gel. The gel plate was placed into the horizontal electrophore- 7

7 sis cell (the sample side should be placed on the negative side). 4-6 layers of gauze were put on each end of the gel. Electrophoresis was performed in borate buffer (0.3 mol/l boracic acid, 0.06mol/L NaOH, ph9.0)at 6 V/cm for 2-3 hours. Staining and Rinsing After electrophoresis, the red bands on the gel was observed directly, and then the gel was put into Ponceau red staining solution for at least 4hours. The gel was rinsed with rinsing solution repeatedly until the background was clean. After drying the gel in the air or in the oven, the gel was stained with Benzidine staining solution(note: Benzidine is highly carcinogenic) for about 20 mins until the Hb bands were stained blue black. The gel was rinsed with rinsing solution, and then scaned under white light using a Gel imaging system. Relationship between HRT and Clinical Disease Erythrocyte Disease and Abnormal HRT The results of HRT showed that the re-released Hb was increased significantly in both β-thalassemia (Figure 1A) and α-thalassemia(figure 1B) patients [3,7]; however the re-released Hb was decreased significantly in hereditary spherocytosis (HS) patient [7]. Thalassemia is caused by the decreased or defective synthesis of β-globin or α-globin chain. The excessive free globin chains are kept 8

8 in intact structurally. However, the erythrocyte membrane can be damaged either by globin chain sedimentation or oxidative damage. The mechanism of abnormal HRT was speculated to have relationship with the abnormal sedimentation of Hb on the erythrocytes membrane [5]. HS is an autosomal dominant erythrocyte membranopathy, which is caused by genetic mutations of membrane proteins, including spectrin (α and β), ankyrin, band 3, protein 4.2 and other erythrocyte membrane proteins. In this study, the decreased Hb re-release during HRT is speculated to have relationship with the decreased interaction between mutated membrane proteins and Hb [7]. Figure 1: Single-direction HRT of thalassemia and hereditary spherocytosis patients(hs). A. Single-direction HRT of β-thalassemia patients. Samples 4 and 6 were blood from two β-thalassemia patients, and the other samples were from normal healthy adults. B. Singledirection HRT of HS and α-thalassemia patients. B represents whole blood sample; R represents erythrocyte sample. Carcinoma Patients and Abnormal HRT There-released Hb from erythrocytes and whole blood of seven carcinoma patients were studied by HRT 9

9 [8]. The results showed that the re-released Hb was distinctively increased from an intra hepatic bile duct carcinoma patient during single-direction isotonic HRT (Figure 2). Further mechanism research work can not be continued because of the patient s leave, but HRT is speculated to have relationship with the abnormally elevated serum bilirubin, which lead to the abnormal sedimentation of Hb on the membrane. Figure 2: One-direction ERT of seven upper gastrointestinal carcinoma patients. There were seven samples; each sample was divided into whole blood group (W) and erythrocyte group (R) correspondingly. Sample 1 and 7 were cardia carcinoma patients; sample2, 3, 4, 5 and 6 were hepatocellular carcinoma patients. Uremia Patients and Abnormal HRT HRT was used to study the re-released Hb from uremia patients. The results showed that in comparison with normal control, the re-released Hb from five chronic renal 10

10 failure (CRF) patients was increased significantly (Figure 3) [9]. Change in the re-released Hb may be due to the elevated serum toxin on the erythrocyte membrane, and lead to the abnormal sedimentation of Hb on the membrane. Figure 3: One-direction hypotonic HRT of uremia patients. Sample 1, 3, 5, 7 and 9 were normal adult patients; sample 2, 3, 4, 6, 8 and 10 were uremia patients. General Surgery Patients and Abnormal HRT One-direction 37 C hypotonic HRT was performed to observe the re-released Hb from general surgery patients. There were ten different general surgery patients, including gallstone, abdominal trauma, intestinal obstruction, papillary cyst cancer, spleen rupture, suppurative appendicitis and pancreatitis [10]. The results showed that the re-released Hb was increased from sample1, 4, 6, 8 and 9 (Figure 4). According to the clinical detection of 11

11 these patients, HRT can be preliminary speculated to have relationship with abnormal lipid metabolism, abnormal hepatic function, poor nutrition and chronic inflammation. However, there are so many affecting facts, and their relationships with HRT were unknown. Figure 4: One-direction hypotonic HRT of general surgery patients at 37 C. Patient 1 to 4 was gallstone; patient 5 to 10 was abdominal trauma, intestinal obstruction, papillary cyst cancer, spleen rupture, suppurative appendicitis and pancreatitis respectively. The re-released Hb was increased from sample1, 4, 6, 8 and 9. Mechanism of HRT The above studies have shown that, in addition to erythrocyte disease, the change of HRT appears in many other diseases. These changes seem to be not only related 12

12 to the disease, but also to the progress of the disease and the occurrence of complications. To explore the mechanism of HRT, some in vitro experiments were performed. The Effects of Oxidative Stress on Erythrocyte HRT In order to study the effects of oxidative stress on HRT, the erythrocytes were treated with different concentration of H 2 O 2 for 1 hour. The HRT results showed that with increasing concentration of H 2 O 2, the re-released Hb was increased significantly (Figure 5) [11]. Figure 5: The effects of oxidative stress on erythrocyte HRT. Erythrocytes were treated with 0, 0.125%, 0.25%, 0.5% H 2 O 2 for 1 hour, H represents hemolysate of normal erythrocytes. 13

13 Effect of Glutaraldehyde on Erythrocyte HRT To find out whether the re-released Hb comes from membrane-bound Hb, erythrocytes were treated with different concentration of glutaraldehyde. The results showed that the re-released Hb was decreased with increasing concentration of glutaraldehyde (Figure 6) [12]. Glutaraldehyde is a bifunctional cross-linking agent, which has two reactive aldehyde groups, and can react with amino, mercapto and other groups. When glutaraldehyde was added into the erythrocytes, the membrane-bound Hb would be cross-linked with the membrane gradually, and the re-released Hb would be decreased with the increasing concentration of glutaraldehyde. When the erythrocytes were fixed entirely, no more Hb would be re-released. Figure 6: Effect of glutaraldehyde on erythrocyte HRT. H represents hemolysate of normal erythrocytes; sample 1 and 7 are normal control; sample 2-6 are erythrocytes treated with %, 0.005%, %, 0.01%, % and 0.025% glutaraldehyde for 1 hour respectively. 14

14 Effects of Plasma Components on Erythrocytes HRT To clarify if the plasma components have effects on the re-released Hb from erythrocytes, HRT was performed to study the effects of different plasma components including albumin, globulin, glucose, amino acid, vitamin, insulin and inorganic ions on re-released Hb from erythrocytes and whole blood samples during HRT. The results showed that albumin, globulin (Figure 7A), amino acid (Figure 7B), vitamin C (Figure 7C), insulin (Figure 7D), NaCl, KCl, CaCl 2 and NaHCO 3 (Figure 7E) reduced the re-released Hb; while glucose (Figure 7F), vitamin B 1, vitamin B 2, and vitamin B 12 (Figure 7C) had the opposite effects on the re-released Hb [13]. These results told us that any change of plasma components can alter the amount of re-released Hb from erythrocytes. 7 A 15

15 7 B 7 C 16

16 7 D 7 E 17

17 7 F Figure 7: Effects of plasma components on erythrocytes HRT. A is the effect of plasma protein (albumin and globulin) on re-released Hb; B is the effect of amino acids on re-released Hb; C is the effect of soluble vitamin on re-released Hb; D is the effect of insulin on re-released Hb; E is the effect of inorganic ions on re-released Hb; F is the effect of glucose on re-released Hb. Conclusion The above preliminary results show that the amount of re-released Hb depends on the interaction between erythrocytes membrane and Hb, abnormalities of membrane protein, Hb and plasma environment can lead to 18

18 the change of re-released Hb during HRT. The sensitivity of various HRT is different: hypotonic HRT is higher than isotonic HRT, 37 C HRT is higher than room temperature HRT. Different levels of sensitivity may reveal different serious of disease. When HRT is performed at room temperature, the re-released Hb from most of the clinical specimens is not increased. Once the re-released Hb is increased significantly, the disease may become more serious, so it has potential clinical application prospect. More work is needed in mechanism research, and more interaction may be found between the red cell membrane and Hb or, the red cell membrane and plasma environment. Acknowledgment This work was supported by grants from Natural Science Foundation of China ( ), Natural Science Foundation of Inner Mongolia (2016MS0801), Science and Technology Foundation of Baotou (2015C ) and Doctoral scientific research foundation of Baotou Medical College. We also especially acknowledge all of the people who donated their blood samples for our research. Reference 1. Smithies O. Zone Electrophoresis in Starch Gels: Group Variations in the Serum Proteins of Normal Human Adults. Biochem J. 1955; 4: Chalvardjian A. Agarose-Starch Gel Electrophoresis of Rat Serum Lipoproteins. J Lipid Res. 1971; 3:

19 3. Scott AC, Fowler JC. Electrophoretic Typing of Glyoxalase I (GLO I) isoenzymes using a mixed starch/agarose gel. Forensic Sci Int. 1982; 3: Qin WB. Electrophoresis Release of Hemoglobin form Living Red Blood Cells. Scientia Sinica Vitae. 2011; 41: Su Y, Shao G, Gao L, Zhou L, Qin L, et al. RBC electrophoresis with discontinous power supply - a newly established hemoglobin release test. Electrophoresis. 2009; 17: Su Y, Gao L, Ma Q, Zhou L, Qin L, et al. Interactions of hemoglobin in live red blood cells measured by the electrophoresis release test. Electrophoresis. 2010; 31: Su Y, Ma HJ, Zhang HW, Gao LJ, Jia GR, et al. Comparative Study of the Amount of Re-released Hemoglobin from α-thalassemia and Hereditary Spherocytosis Erythrocytes. Inherited Hemoglobin Disorders. Croatia: InTech Su Y, Han L, Gao L, Guo J, Sun X, et al. Abnormal increased re-released Hb from RBCs of an intrahepatic bile duct carcinoma patient was detected by electrophoresis release test.biomed Mater Eng. 2015; 26: S

20 9. Gao YQ, Wang CL, Yan Su, Qin LY, Qin WB. A preliminary study on hypotonic hemoglobin release test in patients with uremia. Journal of Clinical and Experimental Medicine. 2010; 9: Han LH, Yan Su, Gao YQ, Gao LJ, Qin LY, et al. Journal of Clinical and Experimental Medicine. 2009; 7: Wei CH, Su Y, Li XJ, Du XH, An L. Effect of different concentrations of hydrogen peroxide on hemoglobin release of erythrocytes. Progress in Veterinary Medicine. 2014; 3: Wei CH, Su Y, Yang L, Li XJ. Effect of Different Concentrations of glutaraldehyde on Hemoglobin Release of Red Blood Cells. Progress in Veterinary Medicine. 2014; 2: Wang CL, Liu LP, Han LH, Su Y, Qin WB, et al. The effect of plasma components on hemoglobin release test in red blood cells (RBC). Toxicol Environ Chem. 2017; 3: