Original research articleOptimization of the method for α-l-fucosidase, β-d-galactosidase and β-d-glucuronidase determination in serum from hemolyzed blood
Introduction
The activity of lysosomal exoglycosidases present in all cells and body fluids, with the exception of erythrocytes [1], reflects the degradation intensity of: glycoproteins and glycolipids (α-l-fucosidase - FUC) [2], glycoproteins, glycolipids and keratan sulfate (β-d-galactosidase - GAL) [3] and glycosaminoglycans (β-d-glucuronidase – GLU) [4,5]. Therefore, determining the activity of particular exoglycosidases in the body fluids may be used for diagnosiing and monitoring the diseases that involve increased catabolism of appropriate glycoconjugates [[6], [7], [8], [9], [10], [11]]. The available research recommend using increased serum GAL activity as a marker of colon [12] and larynx cancers [13]. Serum GLU activity could be a useful marker of hepatitis progression [14] and increased degradation of proteoglycans in diabetes [15]. The increase in serum FUC in patients with liver cirrhosis and small tumors of hepatocellular carcinoma is a promising marker of both aforementioned diseases, especially since the currently used markers (α-fetoprotein and des-γ-carboxy-prothrombin) seem to be less specific in detecting hepatocellular carcinoma than previously expected [[16], [17], [18], [19]].
Determination of serum exoglycosidases, based on quantification at 410 nm, of released 4-nitrophenol from appropriate artificial substrates may be strongly affected by the presence of hemoglobin released by damaged erythrocytes, which show maximum absorbance at 415, 540 and 570 nm [20].
Hemolysis is responsible for rejecting nearly 60% of serums in routine diagnostic laboratories [21,22]. Hemolysis may result from pre-laboratory errors: improper collecting, processing, or transporting of the blood samples to the laboratory [20]. A serious problem arises with in vivo hemolysis that occurs in forensic medicine [23] and genetic diseases, such as: sickle cell anemia [24], autoimmune hemolytic anemia [25], beta-thalassemia [26] and hemolytic uremic syndrome [27], preeclampsia [28], and paroxysmal nocturnal hemoglobinuria [29].
The aim of the present study was to optimize and validate the Marciniak et al. method [5] for determining GAL, GLU and FUC in serum from hemolyzed blood, based on our previous experience [30,31].
Section snippets
Ethics
The research was approved by the Ethics Committee at Lomza State University of Applied Sciences, Łomża, Poland (protocol: 4/2013/13.11.2013).
Materials
Substrates and 4-nitrophenol were from Sigma, St. Louis, MO, USA. We used the following substrates: 4-nitrophenyl-β-d-galactopyranoside (6.7 mM in 0.1 M phosphate-citrate buffer pH 4.3) for GAL determination; 4-nitrophenyl-α-l-fucopyranoside (6.7 mM in 0.1 M phosphate-citrate buffer pH 4.3) for FUC determination; 4-nitrophenyl-β-d-glucuronide (6.3 in 0.1 M
Results and discussion
This paper concerns FUC, GAL and GLU activities in serum from hemolyzed blood based on the determination of released 4-nitrophenol concentration at 410 mn. Determining the 4-nitrophenol concentration is considerably affected by the presence of hemoglobin [20]. The upper limit of the reference range for serum free of hemoglobin is 0.05 g/dL. When hemoglobin serum concentration is above 0.2 g/L, the serum has a visible pink hue, and above 0.6 g/dL, it is distinctly red [33]. Our serums from
Conclusion
Our modification of the Marciniak et al. method [5] make precise determination of FUC, GAL and GLU activities possible for the cases where it is impossible to obtain serum without hemolysis (e.g. genetical diseases, post mortem examinations) or when the blood is hemolyzed during pre-laboratory procedure.
Conflict of interest
The authors declare no conflict of interests.
Financial Disclosure
This work was supported by the Lomza State University of Applied Sciences, Lomza, Poland (protocol number: BST-2/IM/11/2014).
Acknowledgement
We are grateful to Michael Gilmor (BS in biochemistry) from Huntington, New York, USA, for critical reading of the manuscript.
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