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: firstname.lastname@example.org
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
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 
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 . 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)  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 . The chemical contamination of Lake Faro, Italy, was assessed by Cappello T., 2014  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.) , but also mussels (genus Mytilus) .
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 .
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 .
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 , 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
phosphate buffer 0.01M pH7
||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|
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 .
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.|
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|
|Activation factor %|
|Pb(NO3)2 0.001% **||712||–||1204||–|
*Extract ratio: ionic solution tested = 1: 1; ** Extract ratio: ionic solution tested = 3: 1
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.
- Roşoiu, N. (2010). Biochimie medicală, Ed. Ovidius University Press, pp. 626
- Mihele, D. (2006). Biochimie clinică, Ediţia a II-a, Ed. Medicală, pp.486
- Brichacek, L. Allison., Brown M., Candice (2019). Alkaline Phosphatase: A Potential Biomarker for Stroke and Implications for Treatment. Metab Brain Dis. 34(1), pp. 3–19
- Bonaventura, A., Liberale, L., Vecchie, A. et al. (2016). Update on Inflammatory Biomarkers and Treatments in Ischemic Stroke. International Journal of Molecular Sciences 17
- Gandolfi, M., Smania, N., Vella, A. et al. (2017). Assessed and Emerging Biomarkers in Stroke and Training-Mediated Stroke Recovery: State of the Art. Neural Plasticity 2017.pp 15
- Shimizu, Y., Imano, H., Ohira, T. et al. (2013). Alkaline Phosphatase and Risk of Stroke Among Japanese: The Circulatory Risk in Communities Study (CIRCS). Journal of stroke and cerebrovascular diseases 22, pp. 1046–1055
- Liu, J., Wang, D., Li J, et al. (2016). High Serum Alkaline Phosphatase Levels in Relation to Multi-Cerebral Microbleeds in Acute Ischemic Stroke Patients with Atrial Fibrillation and/or Rheumatic Heart Disease. Current Neurovascular Research 13, pp. 303–308
- Gdara, NB., Belgacem, A., Khemiri, I. et al. (2018). Protective effects of phycocyanin on ischemia/reperfusion liver injuries. Biomedicine & Pharmacotherapy 102, pp. 196–202
- Presbitero, A., Mancini, E., Brands, R. et al. (2018). Supplemented Alkaline Phosphatase Supports the Immune Response in Patients Undergoing Cardiac Surgery: Clinical and Computational Evidence. Front Immunol, 9, pp 2342
- Rui Xiao, Li-Ping Xie, Jing-Yu Lin, Qing-Xi Chen, Hai-Meng Zhou, Rong-Qing Zhang (2002) Purification and enzymatic characterization of alkaline phosphatase from Pinctada fucata. Journal of Molecular Catalysis B: Enzimatic, Volumul 17, Numărul 2,pp. 65-74
- Nandurkar, H. P. (2017). Change in Alkaline Phosphatase Activity Induced By Tetracycline in Freshwater Mussel, Parreysia cylindrica (Annandale and Prashad). Journal of Pharmacy and Biological Sciences, Volume 12, Issue 3 Ver. VI, pp. 92-94
- Rob L. Dean. (2002). Kinetic studies with alkaline phosphatase in the presence and absence of divalent inhibitors and cations Biochemistry and Molecular Biology Education, Vol 30, nr 6, pp. 401-407
- Robu, B., Jitar, O., Teodosiu, C., Strungaru, S., Nicoara, M., Plavan, G., (2015), Environmental impact and risk assessment of the main pollution sources from the romanian Black Sea coast, Environmental Enginering Management Journal, Vol.14, No. 2, pp. 331-340
- Mititelu, M., Moroşan, E., Neacșu, S. M., Ioniţă, E. I. (2018). Research regarding the pollution degree from romanian Black Sea coast. Farmacia 66(6), pp. 1059-1063
- Cappello, T., Maisano, M., Parrino, V., Giannetto, A., Brundo, MV. (2014). Cellular biomarkers in the mussel Mytilus galloprovincialis (Bivalvia: Mytilidae) from Lake Faro (Sicily, Italy). Italian Journal of Zoology, Vol. 81, Nr 1, pp. 43-54
- Brent, L., Lockwood, George N. (2012). SomeroFunctional Determinants of Temperature Adaptation in Enzymes of Cold- versus Warm-Adapted Mussels. BME vol. 29, nr. 10, pp. 3061–3070
- Verginica Schröder, Mariana Arcus, Andreea Hortense Anghel, Florica Busuricu, Anca Cristina Lepadatu (2019). Cell differention process of Artemia sp. larvae tools for natural products testing. Scientific Papers. Series D. Animal Science, Bucharest, Vol. LXII, No.1, pp. 149-153
- Bușuricu, F., Schroder, V., Margaritti, D., Nadolu, D., Anghel , A.H.(2019). Preliminary study regarding sodium benzoate and other food dyes sinergic action using BSLA citotoxicity test – Scientific Papers. Series D. Animal Science. Vol. LXII, No. 1, pp. 410-416
- Balaban, D.P., Natalia, R., Busuricu, F., Sava, D. (2004). Etude preliminaire concertant la separation d’un melange d’alginates de l’algue brune Cystoseira barbata. Rapp. Comm. Int. Mer. Medit 37, pp. 485
- Buşuricu, F., Zamfirescu, S., Bioactive Principles isolated from Trygon pastinaca Sea Fish; Composition and Skin Tolerance, J. Med. Biochem, 1999, 3, 3, 227-282
- Buşuricu, F., Roşoiu, N. (2001). Etude des phospholipides de quelques especes d’invertébrés et de poissons de la mer noire, rapp. Comm. Int. Mer. Medit., 36, pp. 185
- Mititelu, M., Ioniţă, A. C., Moroşan, E. (2014). Research regarding integral processing of mussels from Black Sea. Farmacia, 62(3), pp. 625-632
- Buşuricu, F., Popescu, A., Balaban, D.P., Negreanu Pârjol, T., Zamfirescu, S. (2008). Evaluation of the photoprotective action of active principles obtained from Trygon pastinaca, Roum. Biotechnol. Lett. 13, 6, supliment, pp.26-31
- Buşuricu, F., Balaban, D.P., Stanciu, G., Anghel, A. Introductory study regarding the optimization of a new qualitative method used for checking the milk pasteurization. Ovidius University Annals of Chemistry, Vol.18, Nr. 1, pp. 17-21
- IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37°C. Part 9. Reference procedure for the measurement of catalytic concentration of alkaline phosphatase. Clin Chem Lab Med 2011; 49:1439-144