Valorification of Mussels Mytilus galloprovincialis and Rapana thomasiana, from the Coast of the Romanian Black Sea Coast, for Obtaining Alkaline Phosphatase (AP)

BUȘURICU Florica1, SCHRÖDER Verginica1*, MITITELU Magdalena2, IONIȚĂ Ana Corina2, NICOLESCU Teodor Octavian2, NICOLESCU Florica2

1Faculty of Pharmacy, Ovidius University, Mamaia Blvd. 124, 900527, Constanta (ROMANIA)

2Faculty of Pharmacy, ”Carol Davila” University of Medicine and Pharmacy, 6, Traian Vuia Street, 020956, Bucharest (ROMANIA)

 *Corresponding author: virgischroder@yahoo.com 

Abstract

     For the clinical analysis and biological research, commercially available preparations containing bovine intestinal alkaline phosphatases and those of bacterial nature are used. Marine organisms are recognized for their nutritional and therapeutic values, some being an important source of alkaline phosphatase (AP).

     In this paper we shall present data on the optimal conditions for obtaining the enzyme extract with alkaline phosphatase activity, from 2 marine organisms – Mytilus galloprovincialis and Rapana thomasiana, from the Black Sea, the Romanian coast, as well as the influence of metal ions on enzymatic activity.

     Considering that obtaining the enzyme extract does not involve high costs and that these marine organisms are sufficiently available, the importance of the study lies in the applicability of AP exploration as an alternative in treatments and clinical analyzes.

Keywords: alkaline phosphatase, Mytilus galloprovincialis and Rapana thomasiana, enzyme activators and inhibitors

Introduction

     Alkaline phosphatase (AP) or phosphate monoester hydrolase is a metalloenzyme, having in its structure the zinc ion. The biological sources in which it predominates are: liver, bones, intestine, placenta and white blood cells. Along with phosphorus and calcium, two mineral elements essential for bone mineralization, alkaline phosphatase participate in the development of the bone system and cartilage, being a specific marker of osteoblasts [1, 2].

     In clinical practice, the standard alkaline phosphatase test represents the sum of all the activity of soluble isoenzymes in serum or plasma, reporting the total activity of the enzyme and not the amount of total proteins [2]

    Elevated values of alkaline phosphatase in the blood are found in diseases of the skeletal system (Paget’s disease, rickets), hyperparathyroidism and liver damage. The intensity of the AP increase reflects the magnitude of the respective dysfunction, the increases being 3- 10 times above the upper limit of normal [1, 2]. Often, serum increases in this enzyme occur as a result of the body’s exposure to X-rays or as a response of the body to some drug treatments [1]. Low blood levels of AP may indicate: severe hepato-biliary dysfunction (intra- and extra-hepatic bile cholestasis; biliary cirrhosis, liver neoplasm), hypophosphatasia, leukemia, pernicious and aplastic anemia, malnutrition [1, 2, 3].

    A number of biomarkers are known and used to determine and differentiate stroke (myokines, cytokines, chemokines, neuropeptides, immune cells, growth factors) [4, 5]. To increase the ability to diagnose and predict this disease, new therapies are being sought. Recent clinical trials highlight the possible use of AP as a diagnostic and prognostic marker for stroke [6, 7, 8]. Patients undergoing cardiothoracic surgery with severe inflammation are treated with alkaline phosphatase in current pharmaceutical forms. The results of the studies show the protective cascading effect of supplemented AP in these patients, improving the neurological signal, limiting inflammation and promoting vascular homeostasis [3, 9].

     For clinical analysis and biological research, commercially available preparations containing bovine or calf intestinal alkaline phosphatases and those of a bacterial nature are used. The interest in increasing the range of these enzyme preparations has led to the expansion of research to be obtained from other sources, such as milk (the only food in which this enzyme is found) [10] or marine organisms [11, 12].

     Many marine organisms (algae, molluscs, crustaceans and fish) are used as bioindicators in European / global pollution monitoring programs to assess the impact and risk induced on the marine environment of pollution, in particular by heavy metals, polycyclic aromatic hydrocarbons (HPA), polychlorinated bisphenols (PCBs) and organophosphorus compounds (POs). Most studies are performed using the most common species of mollusks – mussels – Mytilus galloprovincialis. Thus, Robu B. and all., 2015 and Mititelu M., 2019 [13, 14] use this mussel to determine the bioconcentration of heavy metals in the Black Sea, along the Romanian coast, trying to calculate the estimated rate of ingestion / uptake of metals by human consumption of fish [13]. The chemical contamination of Lake Faro, Italy, was assessed by Cappello T., 2014 [15] following on the Mytilus galloprovincialis the statistically significant inhibition (p <0.0001) of digestive gland exposure biomarkers (cytochrome P450 4 and CYP4) and gill neurotoxicity parameters (acetylcholinesterase, AchE and choline acetyltransferase, ChAT). Metabolic and oxidative stress, due to changes in the aquatic ecosystem, due to anthropogenic activities, has been analyzed by many other researchers, using cultures of green algae (Ulva sp., Cladophora sp., Enteromorpha sp.) and red algae (Ceramium sp.) [13], but also mussels (genus Mytilus) [16].

     Some aquatic crustaceans are biotest organisms, such as Artemia salina and Gammarus balcanicus, used for in vivo assessment of the toxicity of certain chemical compounds or the biological effect of an active principle on the human body [17, 18]. In the study of the Romanian marine research, a series of Romanian researchers stood out, capitalizing nutritionally and therapeutically, the marine organisms from the Romanian marine area [19, 20, 21, 22]. Some of the personal research previously performed on marine organisms, have also highlighted the photoprotective effect of some active principles in sea catfish – Trygon pastinaca [23].

Objective

     In this paper, we present current data, obtained at a reassessment of the enzymatic potential in alkaline phosphatase, of the extracts obtained from Mytilus galloprovincialis and Rapana thomasiana, as well as the dynamics of the influence of some metal ions on the enzymatic activity; mussels are harvested from the coast of the Romanian Black Sea coast, Jupiter resort, in the period 2018-2019. Given that the enzymatic and nutritional potential of marine organisms may change due to anthropogenic activities, we made a comparison of current results with those in 2004-2006 [24].

     To obtain an optimal extract with high enzymatic activity, we tested the optimal extraction and incubation conditions following the following parameters: extraction environment, time and temperature, respectively, pH and incubation temperature of the enzyme. Establishing these optimal conditions, we further followed, such as the enzymatic activity influenced by the bivalent ions Mg2+, Co2+, Mn2+,   Zn2+, Cd”+, Pb2+ and Hg2+.  

Material and Methods

     Marine organisms are harvested from the coast of the Romanian Black Sea coast, Jupiter resort, between March and September 2018-2019. The whole body of Mytilus galloprovincialis and the muscle of Rapana thomasiana mollusk were crushed with the 15 mL ROTH borosilicate glass homogenizer, using the ratio of 1 gram to 10 mL extraction medium.

      Enzymatic activity was determined by the colorimetric method [25], using ByoSistems kits, having as substrate 10 mM p-nitrophenylphosphate. The samples are incubated for 30 minutes at a temperature of 37o C, and the absorbances are read at the λ = 405 nm. One unit of enzymatic activity is the amount of alkaline phosphatase enzyme that catalyzes the conversion of one micromole of substrate per minute. To calculate specific enzymatic activity, proteins were determined using the standard 70g / L albumin ByoSistems kit

      We performed the extraction in the phosphate buffer solutions: 0.01M Na2HPO4/NaH2PO4 pH 7; Tris buffer 0.01M HCl, pH8; 0.015M MgCl2; 0.15M NaCl; 0.15M CaCl2; 0.15M KCl; 0.15M sodium acetate. The modification of the ionic activity by the bivalent ions was performed using the solutions: MgCl2 2%, CoCl2 2%, CoSO4 2%, MnCl2 2%, ZnCl2 2%, CdCl22%, Pb (NO3) 2 0.001% mg / mL, Hg (NO3) 2 0.1N. All determinations were performed multiplied by 3 times.

Results and Discussions

Optimal extraction conditions

     To determine the optimal extraction conditions for proteins with alkaline phosphatase activity, we tested several extraction media, finding that the pH 7 phosphate buffer selectively extracts a higher amount of protein. The extract from Mytilus galloprovincialis has an optimal enzymatic activity equal to 350 U/L / min, respectively 97.22 U/g/min (Table 1). As AP was found to be best extracted in 0.01 M phosphate buffer pH 7, we tested the extraction time and temperature of extraction (Table 2).

Table 1. Enzymatic activity at pH 9 and 200C in Mytilus galloprovincialis

No.crt Extraction medium Protein concentration

mg/mL

 Enzymatic activity
mU/mL/min mU/mg/min
Distilled water 1.78 60.15 33.79
0.15M NaCl 1.03 40.35 39.17
0.15M KCl 0.93 36.89 39.67
0.15M CaCl2 1.52 32.66 27.04
0.01MgCl2 2.10 47.42 22.58
CH3COONa 0.15M 1.22 53.45 48.59
Na2HP4 /NaH2PO4

phosphate buffer 0.01M pH7

 3.6 350.00 97.22
0.01M Tris HCl buffer 1.29 33.54 26.00

 

Table 2. Determination of the optimal extraction time and temperature of

alkaline phosphatase from Mytilus galloprovincialis

No. crt. Extraction time (h) Enzymatic activity at 40C Enzymatic activity at 200C
mU/mL/min mU/mg/min mU/mL/min mU/mg/min
1 ½ 175.34 269.75 137.00 152.22
2 1 462.34 355.64 350.00 218.75
3 2 260.50 191.54 215.32 341.77
4 24 150.25 103.62 89.53 63.04
5 48 60.32 92.80 30.36 33.36

 

     We found that at 40C, for 1 hour, the most is extracted, corresponding to an enzymatic activity of 460.34 mU/mL/min, respectively a specific enzymatic activity of 355.64 mU/mg/min, compared to the lower values obtained at a temperature of 200C. The extraction time ranged from 1 hour to 48 hours, and it was found that as time increased by more than 1 hour (time limit proved to be the most efficient) the amount of protein extracted increased, with possible other hydrolase activities (being amylases and proteases), but alkaline phosphatase loses its activity over time.

Optimal incubation conditions

      Establishing the optimal extraction conditions, even if we tested them only on Mytilis galoprovincialis, allows us to further extrapolate them to Rapana thomasiana. Therefore we continued testing the influence of incubation time and temperature on the solubilization of alkaline phosphatase, for both sources of AP. The results show a maximum activity at a pH between 9.8-10.4 and a temperature between 30- 400C (Tabel 3). Of the two sources, Rapana thomasiana – 1288 IU/L has higher enzymatic activity, compared to Mytilis galoprovincialis – 792 IU / L. Comparing the current values obtained with those performed by the undersigned during 2004-2006, there are small differences, the protein and implicitly enzymatic potential of mussels being on average constant [24].

Table 3. Optimal alkaline phosphatase incubation parameters from

Mytilis galoprovincialis and Rapana thomasiana

Determining the optimal pH Determining the optimal temperature
pH Enzymatic activity UI/L Temperatura 0C Enzymatic activity UI/L
Mytilis g. Rapana th Mytilis g. Rapana th.
8.0 230 352 20 400 555
9.0 560 869 25 430 886
9.8 785 1230 30 785 1230
10.4 792 1288 40 792 1288
11.5 416 645 60 462 690

Influence of bivalent ions on enzymatic activity

     The activity of an enzyme can be activated or inhibited by a number of compounds with which it can interact. We chose to add to the extraction medium, after a prior purification of the enzyme, the following bivalent ions: Mg2+, Co2+, Mn2+, Zn2+, Pb2+, Cd2+ and Hg2+. The results obtained (Table 4) show that the ions Mg2+, Co2+, Mn2+ activate the enzyme, while Zn2+, Pb2+, Cd2+, Hg2+ inhibited the enzyme. The influence of these ions, similarly, less for Zn2+, Pb2+, was also observed for the AP obtained from the mussels of other researched marine areas [10, 12]. The order of the ions that increased the enzymatic activity is: Mg2+˃ Co2+ ˃ Mn2+, and that of those that decrease is: Cd2+˂ Zn2+.  Pb2+ and Hg2+ ions do not alter enzymatic activity (Table 4), and cobalt ions in the form of sulfate activate more than the form of cobalt chloride.

Table 4. Influence of bivalent ions on alkaline phosphatase

Ion test solution Mytilis galoprovincialis  Rapana thomasiana
Enzymatic activity

UI/L

 Activation

factor %

 Enzymatic activity

UI/L

 Activation factor %
Extract standard 720 – 1186
MgCl2 2%* 1260 175 2004 169
CoSO4 2%* 1181 164 1898 160
CoCl2 2%* 950 132 1660 140
MnCl2 2%* 1037 144 1613 136
ZnCl2 2%* 547 76 827 70
CdCl2 2%* 590 82 1032 87
Pb(NO3)2 0.001% ** 712 – 1204 –
Hg(NO3)2 0.1N** 708 – 1150 –

*Extract ratio: ionic solution tested = 1: 1; ** Extract ratio: ionic solution tested = 3: 1

Conclusions

      Considering that currently, for different commercial and research purposes, animal or microbial AP is predominantly used, in the future it is possible to capitalize on the 2 marine sources – Mytilus galloprovincialis and Rapana thomasiana from the Black Sea, the Romanian coast, in order to obtain AP.

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