DIAGNOSTIC VALUE OF INDICATORS OF HIGHER FATTY ACIDS IN EVALUATION OF DEVELOPMENT OF DELAYED CONSOLIDATION OF FRACTURES Miromanov A.M., Mironova O.B., Staroselnikov A.N., Miromanova N.A.
Chita State Medical Academy, Chita, Russia
Currently, the rate of injuries presents one of the main social problems and takes the second place among call for medical care in the Russian Federation. The high proportion of complicated fractures is taken by slow union, which happens in 5-51.8 % of cases according to various authors [1, 2].
Despite the fact that the management of slow fracture union has improved over the last decades, early diagnostics of this complications is still actual and difficult. A lot of authors proved that the main factors, which determine the features of the course of union of long bone fractures, are disordered perfusion in the fracture site, disbalance in immune system and in lipid peroxidation system which are responsible for regeneration of tissues in the body [3-6].
The most important link in pathogenesis of the reviewed abnormal process is lipid peroxidation (LPO), which includes higher fatty acids (HFA). Their role is determined not only by cell energy formation by means of oxidation of substrates of saturated and monoenoic fatty acids, but also by formation of membranes by means of non-saturated fatty acids. Moreover, it has been proved that polyenoic fatty acids take direct participation in the complex mechanism of formation of eicosanoids and aminophospholipids. When metabolism of fatty acids is disordered, an unfavorable influence on links of pathogenesis of insulin resistance syndrome appears [7].
Fatty acids (FA) are essential building material for various tissues of the body (by means of formation of lipid connections), including bimolecular phospholipid layer of cells which is the basis for receptors, various transport systems and enzymes [8]. The important role of lipids consists in the fact that they are predecessors of many biologically active substances participating in various abnormal processes, including ones in disorders of reparative regeneration of bone tissue. Therefore, the disorder of properties of lipid layer can be reviewed as the main cause of a disease or its complications [9, 10].
Considering the above mentioned facts, the definite scientific interest is related to research of qualitative and quantitative composition of fatty acids of serum lipids in disordered fracture union of long bones, other researchers in this field, and identification of diagnostic criteria. It is perspective in theoretical and practical relation.
Objective − determination of the diagnostic significance of higher fatty acids in the development of delayed consolidation in patients with fractures of long bones of the extremities.
MATERIALS AND METHODS
The study was conducted in compliance with ethical principles of World Medical Association Declaration of Helsinki, 1964, 2013, and the Rules for Clinical Practice in the Russian Federation confirmed by the Order of Health Ministry of Russia on June 19, 2003, No. 266.
A retrospective clinical study (case-control) included 30 patients, age of 20-40, with malunion of long bone fractures by the type of slow consolidation. The characteristics and location of fractures corresponded to 41C2 (6.7 %), 42A2 (43.3 %), 42C1 (26.7 %) and 43A1 (23.3 %) according to classification by M.E. Muller et al (1996).
The control group included 20 almost healthy men and women (age of 20-40). The exclusion criteria were acute or chronic abnormal conditions or processes.
The treatment of the patients was realized according to the present national guidelines for traumatology of the Russian Federation [2].
The levels of higher fatty acids (HFA) were measured in peripheral venous blood with use of the common techniques. The quantitative analysis of volatile fatty acids was conducted according to M.D. Ardatskaya. The lipids were extracted with use of the technique by J. Folchetal (1957) to determine the range of HFA (myristic acid – Ñ13Í27ÑÎÎÍ, palmitic acid – Ñ15Í31ÑÎÎÍ, palmitooleic acid – Ñ15Í29ÑÎÎÍ, stearic acid – Ñ17Í35ÑÎÎÍ, oleic acid – Ñ17Í33ÑÎÎÍ, linoleic acid – Ñ17Í31ÑÎÎÍ, α-linoleic – Ñ17Í29ÑÎÎÍ, γ-linoleic – Ñ17Í28COOH, dihomo-γ-linoleic – Ñ19Í33ÑÎÎÍ and arachidonic acid – Ñ19Í31ÑÎÎÍ). Then the HFA aliquot was evaporated and methylated according to K.M. Sinyak et al (1976). Methyl esters were purified in chromatographic system − in thin layers of silica gel hexane : diethyl ester: glacial acetic acid (90 : 10 : 1). The next stage was their extraction with mixture of chloroform : methanol (8 : 1). The analysis was conducted with the chromatograph Crystal-2000M (Russia) with plasma ionisation detector and capillary column 0.35 × 50 FFAP (USA). Analitika hardware complex was used for calculation and determination of peaks [11].
The instrumental study (X-ray imaging) of the leg was conducted in direct and lateral plains before and after surgical intervention after 1, 2, 3 and 6 months from the surgery. The radiologic signs of complete union of a fracture: continuous and smooth calcification of callus with higher density, union and absorption of external callus, continuous beams between bone fragments. The signs of fracture union were estimated with RUST (Radiographic Union Scale for Tibial Fractures) (B.W. Kooistra et al., 2010). Complete fracture union was at the sum of 10 and more points (the table 1) [12].
Table 1
X-ray score for estimation of fracture consolidation
|
Absolute value* |
1 |
2 |
3 |
|
Callus |
No |
Yes |
Yes |
|
Fracture line |
Visible |
Visible |
Not visible |
Note: * – a digital value is calculated for each edge of cortical layer of a bone (anterior, posterior, medial, lateral) in fracture site; no consolidation – 4 points; complete consolidation – 12 points.
The received results were analyzed with BIOSTAT software. Before analysis, the variations sets were tested for normalcy by means of skewness and excesses. The median (Me), and 25 and 75 percentiles (P25-P75) were calculated with descriptive statistics. Mann-Whitney test was used for comparison of two non-related groups. P value ≤ 0.05 was considered as statistically significant.
RESULTS AND DISCUSSION
Patients with slow union of long bone fractures showed some transformations in the spectrum of fatty acids of venous blood lipids (the table 2).
Table 2
Values of higher fatty acids in patients with delayed consolidation, Me [P25 – P75]
|
Molecular formula, systematic name and (systematic formula) of higher fatty acids |
Control group (n = 20) |
Group with delayed consolidation (n = 30) |
|
Ñ13Í27ÑÎÎÍ tetradecanoic (Ñ14:0) |
1.23 [0.85; 1.37] |
1.06 [0.48; 1.15] * |
|
Ñ15Í31ÑÎÎÍ hexadecanoic (Ñ16:0) |
21.68 [17.96; 27.44] |
24.71 [20.45; 28.47] * |
|
Ñ15Í29ÑÎÎÍ cis-9-hexadecenoic (Ñ16:1) |
2.95 [2.07; 4.58] |
2.55 [1.97; 3.4] |
|
Ñ17Í35ÑÎÎÍ octadecanoic (Ñ18:0) |
7.8 [6.56; 10.09] |
5.09 [4.64; 5.66] * |
|
Ñ17Í33ÑÎÎÍ cis-9-octadecanoic (Ñ18:1) |
23.39 [20.69; 25.64] |
24.17 [22.48; 29.05] |
|
Ñ17Í31ÑÎÎÍ cis,cis-9,12-octadecadienoic (Ñ18:2 ω6) |
32.88 [32.46; 35.37] |
31.43 [27.9; 33.46] |
|
Ñ17Í29ÑÎÎÍ cis,cis,cis-9,12,15-octadecatrienoic (Ñ18:3ω3) |
2.28 [1.99; 2.62] |
0.6 [0.34; 1.78] * |
|
Ñ17Í28COOH cis,cis,cis-6,9,12-octadecatrienoic (Ñ18:3ω6) |
0.7 [0.54; 0.82] |
0.26 [0.15; 0.65] |
|
Ñ19Í33ÑÎÎÍ 8,11,14- eicosatrienoic (Ñ20:3ω6) |
1.08 [0.69; 1.48] |
0.22 [0.13; 0.72] * |
|
Ñ19Í31ÑÎÎÍ öèñ-5,8,11,14- eicosatetraenoic (Ñ20:4ω6) |
4.41 [3.03; 5.1] |
3.11 [2.34; 3.82] * |
It was found that tetradecanoic and octadecanoic acids decreased (1.2 and 1.5 times correspondingly), and hexadecanoic acid increased 1.1 times as compared to the control group (p ≤ 0.05).
A decrease in level of polyunsaturated fatty acids by 3.8 times as compared to the control value was noted only for cys,cys,cys-9,12,15-octadecatrienoic acid. A decrease in HFA of ω-6 series was fixed due to reduction of 8,11,14-eicosatrienoic and cys-5,8,11,14-eicosatetraenoic acids by 4.9 and 1.4 times correspondingly (p ≤ 0.05).
It was shown that the main energetic substrate for cells of the macroorganism was fatty acids. Many abnormal processes and conditions are characterized by various disorders in utilization, changes in their level and qualitative composition in the blood serum [7].
A decrease in level of polyunsatutated fatty acids in composition of lipids is characterized by hyperintensification of processes of LPO which was noted by many authors [13].
Moreover, a significant increase in synthesis of eicosanoids in the abnormal process also results in reduction of polyunsaturated fatty acids. The biological effect of higher fatty acids is related to esters and free forms. FAs enter the cells of the body. Acetyl coenzyme A synthetase passes into Acyl-CoA. The latter one enters mitochondria by means of carnitine transferase, where it is exposed to beta-oxidation, and it transforms into acetylCoA, which participates in Krebs cycle with subsequent release of ATP. Due to homeostasis of processes of anabolism and catabolism, the continuous presence of the pool of these substances in tissues is realized. Deficiency or excess of fatty acids make the negative influence on energetic metabolism of cells since disconnection of mechanisms of oxidative phosphorylation and biologic oxidation of FA appears, and activity of mitochondrial enzymes slows down. The function of kalium-sodium pump is disordered. As result, the flow of kalium ions into the cells increases, and the potential of cellular membrane changes. Owing to structural changes in phospholipid layer of cellular membrane, calcium ions show excess in cells, resulting in increasing activity of phospholipase, leading to cellular damage and death [14, 15].
It has been proved that one of the most important processes in developing complications of long bone fractures is intensification of products of lipid peroxidation (also in biological membranes). For example, lipid oxidation is accompanied by hemolysis of red blood cells, and their rheologic properties disorder as result of inevitable injury to membrane structure and disorder of their permeability for ions. In most cases, POL causes the damage of unsaturated FA (linoleic, arachidonic, docosahexaenoic) since they are in composition of biological membranes. A damage causes a change (rotation) of lipid spectrum of biological membranes by means of increasing hydrophilia of their molecules as result of lipid oxidation and formation of peroxides. Moreover, the formed products of POL have the high reactive activity, particularly, products with conjugated double bonds and/or aldehyde groups. It was found that 4-hydroxynonenal (the main product of oxidation of 1,6-arachidonic acid) promotes the mutation and death of cells by means of disorder of structure of protein molecules as result of their ligation and deactivation of enzymes. The destructive activity of 4-hydroxynonenal is realized by means of formation of amino-acid residues of L-α-amino-β-imidasolylpropionic (His), 2,6-diaminohexanic (Lys) and α-amino-β-thyodipropionic (Cys) acids in composition of proteins with covalent adducts [16].
The study supposes that the observed disbalance in FA metabolism makes the negative influence on cells participating in the process of reparative regeneration of bone tissue, resulting in disorder of fracture union.
Therefore, a study of HFA in combination with the known diagnostic criteria of malunion can be the perspective direction in traumatology and orthopedics.
CONCLUSION
Slow union of long bone fractures is characterized by a decrease in serum level of saturated fatty acids - C14:0, C18:0, and an increase - C16:0, whereas the group of unsaturated fatty acids shows a decrease in Ñ18:3ω3, Ñ20:3ω6 and Ñ20:4ω6.
Information on financing and conflict of interests
The study was conducted without sponsorship. The authors declare the absence of any clear and potential conflicts of interests relating to publication of this article.