CC BY
18
UDC 615.254.1:612.63-092.4
INFLUENCE OF FUROSEMIDE ON THE VOLUME AND COMPOSITION OF AMNIOTIC FLUID IN RABBITS ON THE 27-28TH DAY OF PREGNANCY
1 Altai State Medical University, Barnaul
2 OOO "SPF Helix", Saint-Petersburg
Yu.V. Shabalina1, A.V. Popovtseva2, E.V. Suzopov1, Yu.V. Degtyarev1, S.V. Zamyatina, V.M. Bryukhanov1, A.Yu. Zharikov1, Yu.V. Korenovsky1
Research objective: to study the influence offurosemide on the volume and composition of amniotic fluid (AF) of first-pregnant rabbits on the last gestation days.
Material and methods: an experimental (11 pregnant rabbits) and control (6 rabbits) group were used in this study. Volume of amniotic fluid, osmolality and concentration of electrolytes - Na+, K+, Cl~, non-organic phosphate (P), Ca2+ and organic components (creatinine, urea, lactate) in AF were measured.
Results: it was found thatfurosemide caused an increase in volume of AF, as well as an increase in AF's electrolytes (Na+, K+, Cl-, P , Ca2+) and organic (lactate, creatinine) components.
Conclusion: changes in AF parameters are associated with increasing of AF volume, electrolytes (Na+, K+, Cl~, P, Ca2+) and organic (lactate, creatinine) components, which were caused by deceleration of water resorption in fetus kidneys.
Key words: amniotic fluid, pregnancy, fetus, furosemide, rabbits.
Amniotic fluid (AF) is a biologically active fluid that is contained inside the membranes during pregnancy [1]. AF surrounds the fetus and is its natural environment, while playing a significant role in ensuring its life. The most important functions of AF are its role in the process of metabolism of the fetus, as well as the protection of the fetus from all external influences [2]. The safe course of pregnancy depends on the normative state of the amniotic fluid [2].
AF reflects homeostasis of both fetuses and the maternal organism [3]. Determination of the normal biochemical parameters of AF is necessary to study the regulation of its ionic composition, volume and mechanism of education, which will allow the development of a valid model for studying the pharmacokinetics and pharmacodynamics of drugs in pregnant organisms [4]. Much remains unclear in the physiological regulation of the volume of amniotic fluid (AF) and its composition, which is due to two factors: 1) existence of various potential ways of AF formation and outflow (fetal urine, intramembrane pathway, ingestion, lung secretion, nasopharyngeal secretion, transmembrane and percutaneous paths, as well as transfer through the surface of the umbilical cord); 2) the rate of transition of water and solute through most of these paths is rarely measured simultaneously [5].
In the second half of pregnancy, the amniotic fluid presents the fetal urine and the lung secretion [6]. Amniotic fluid is swallowed by the fetus, and also through the amnion enters the bloodstream of the fetus. The amount of amniotic fluid depends on the degree of hydration of the fetus [6]. Since all the fetal water comes from the mother, the placental
flow of water is an important factor determining the volume of amniotic fluid [7].
However, this concept does not explain the mechanism of regulation of the volume of amniotic fluid during pregnancy, the mechanisms for reducing the volume of amniotic fluid during a post-term pregnancy or in acute oligohydramnios [3]. Since fetal urine is the main way to form AF, fetal kidney susceptibility to diuretics, in particular, to furosemide, is of great interest. The results of this study will identify the extent and nature of changes in the volume and composition of amniotic fluid in the model [8].
The purpose of the work was to study the effect of loop diuretic furosemide on the volume and composition of amniotic fluid of rabbits in late pregnancy.
Materials and methods
The clinical trial included 17 primigravida rabbits and their 104 fetuses. Fertilization was carried out by various randomly selected males, after which the rabbits were kept in single cages on a free diet. All animals were divided into 2 groups (experimental and control). The experimental group included 6 rabbits and 31 fetuses. The control group - 11 rabbits and 73 fetuses. Rabbits of the experimental group were injected with furosemide diuretic at a dose of 2.9 mg per kg of body weight. Injection of furosemide was made into the marginal ear vein, which runs along the thin edge of the ear on its outer surface. The wool was cut along the vein. The ear was lightly massaged and wiped with alcohol to create enhanced blood circulation. The vein was clamped at the base of the ear, the ear was taken, and the needle of the syringe was injected
into the cavity of the vessel. Puncture of the veins began closer to the top of the ear. After the vein was punctured, the needle in the vessel was fixed. After that, the squeezing of the vein was finished and, holding the syringe in the right hand, an injection was made. The control group of rabbits did not receive diuretic. The criteria for the inclusion of test animals are: gestational age of 27-28 days (for normal pregnancy - 31 days), mass of the rabbit of 3-4 kg. The criterion for excluding fetuses from research is the weight of the fetus less than 20 g.
During the experiment, the fetus weight, placenta weight, volume and osmolality of the amniotic fluid were determined. In the amniotic fluid, the concentration and content of organic (lactate, creatinine, urea) and inorganic (sodium, chlorine, potassium, calcium and phosphate ions) substances were determined.
For the study, 15 minutes after the injection, cervical dislocation and midline laparotomy with uterus extirpation were performed. Amniotic sacs with fetuses were isolated and removed from the uterine cavity and the maternal and fetal parts of the placenta were separated without disturbing the integrity of the amniotic sac. The amniotic fluid was removed from the amniotic sac by a single-use syringe. Samples of the amniotic fluid were centrifuged for 15 min, placed in 200 ^l microtubes and frozen at -20 ° C until biochemical examination. Biochemical studies were performed on an automatic biochemical analyzer Dimension Xpand (Siemens, Germany). The concentration of Na+, K+ and Cl- ions was determined by a potentiometric method using the QuikLyte Integrate Multisensor module (Siemens, cat. No. S600, USA); total concentration of Ca2+ - by the colorimetric method with CA Calcium Flex reagent cartridge reagent kit (Siemens, Cat. No. EA4164, USA); the concentration of Pi - by a colorimetric method using
In the course of the study of the amniotic fluid composition, inorganic and organic components were determined (Tables 2, 3). In the amniotic fluid of rabbits of the experimental group, the content of ions (sodium, potassium, chlorine, calcium and phosphate) is significantly higher than that of the control group, which indicates the effect of furosemide on the composition of the amniotic fluid. The concentration of ions (sodium, potassium,
a PHOS Phosphorus Flex reagent cartridge reagent kit (Siemens, Cat. No. EA4172, USA). Osmolality of AF was determined using a Vapro osmometer (Wescor, United States). The concentration of creatinine in AF was determined by the modified Jaffe method using CREA Creatinine Flex reagent cartridge reagents (Siemens, cat. No. DA4254, USA); the concentration of urea - kinetic enzymatic (urease) method using BUN Urea Nitrogen Flex reagent cartridge (Siemens, cat. No. EB4309, USA); the lactate concentration was determined by an enzymatic, amperometric method using a glucose analyzer and a "Biosen C line" lactate by EKF (Germany).
The study was approved by the ethical committee of the Altai State Medical University, Ministry of Health of Russia.
By the statistical analysis of the results of the study using the SigmaPlot 11.0 program, the median, 25 and 75 percentiles, the accuracy of intergroup differences according to the MannWhitney test were calculated. The reliability level for all criteria used was taken as p <0.05.
Results and discussion
The mass of the fetus did not affect the selection of groups of animals. Significant differences were observed in the mass of the placenta, volume and osmolality of the amniotic fluid. Furosemide is a loopback diuretic. It violates the reabsorption of sodium ions, chlorine in the ascending part of the loop of Henle. Due to the increased excretion of sodium ions, occurs a secondary (mediated osmotically bound water) enhanced water excretion and an increase in the secretion of potassium ions in the distal part of the renal tubules [9]. Therefore, the introduction of furosemide in the experimental group of animals led to a change in overall indicators. Table 1 presents the characteristics of the study of general indicators.
chlorine, phosphate) does not change, indicating that the urine is diluted. With the introduction of furosemide, the content of ions (sodium, potassium, chlorine, calcium, phosphates) in the amniotic fluid increases, diuresis increases, the speed of urine movement along the kidney tubules increases, indicating insufficient reabsorption of lactate and creatinine. Due to the increased diuresis, the urea content decreases due to dilution of urine.
Table 1
Determination of general indicators (fetal weight, placenta weight, osmolality, volume) of amniotic fluid according to the Mann-Whitney test
Indicator Furosemide Control P
fetus weight, g 24,9; 26,6; 31,3 26,6; 31,0; 34,2 0,108
placenta weight, g 3,9; 4,2; 4,6 2,7; 3,2; 3,7 0,008
amniotic fluid volume, ml 0,4; 3,3; 4,2 0,3; 0,6; 1,0 0,037
osmolality, mosmol/kg 289,0; 303,0; 314,0 223,0; 231,0; 245,0 <0,001
Note: data is presented in the form of 25 percentiles, median, 75 percentiles
Table 2
Determination of inorganic components (ions) in amniotic fluid according to the Mann-Whitney test
Indicator Furosemide Control P
Na, mmol/l 115,0; 138,5; 142,0 129,0 132,0; 135,0 0,325
Na, |jmol 62,5; 496,1; 583,8 43,8; 85,1; 114,40 0,037
K, mmol/l 5,6; 7,5; 20,1 6,5; 7,2; 8,3 0,981
K, |jmol 12,8; 20,3; 28,0 3,06; 4,7; 9,5 <0,001
Cl, mmol/l 94,0; 102,5; 108,0 100,0; 102,0; 106,0 0,981
Cl, |jmol 45,0; 366,1; 432,6 33,9; 67,5; 90,4 0,042
Ca, mmol/l 2,4; 3,6; 5,3 2,04; 2,1; 2,3 <0,001
Ca, |jmol 2,3; 12,7; 14,2 0,6; 1,3; 2,9 0,015
P, mmol/l 1,4; 1,5; 2,1 0,9; 1,0; 1,1 0,010
P, |jmol 2,1; 3,4; 5,6 0,3; 0,7; 1,3 <0,001
Note: data is presented in the form of 25 percentiles, median, 75 percentiles
Table 3
Determination of organic components of amniotic fluid (lactate, creatinine, urea) according to the Mann-Whitney test
Indicator Furosemide Control P
Lactate, mmol/l 16,8; 20,3; 25,4 11,7; 12,3; 15,0 0,003
Lactate, ^mol 9,0; 48,4; 117,3 4,0; 8,0; 18,0 0,009
Creatinine, mmol/l 0,1; 0,1; 0,2 0,1; 0,1; 0,1 0,007
Creatinine, ^mol 0,2; 0,5; 0,8 0,06; 0,09; 0,19 0,001
Urea, mmol/l 4,2; 4,2; 6,5
Note: data is presented in the form of 25 percentiles, median, 75
By determining the organic components of the rabbit amniotic fluid (lactate, creatinine and urea), significant differences were revealed between the experimental and control groups. The lactate content in the control group was 8.0 ^mol, and in the experimental group - 48.4 ^mol, while the creatinine content in the experimental group was 5.5 times higher than the control creatinine content. The urea content in the experimental group of animals is lower than in the control group. This proves the effect of furosemide on the composition of the amniotic fluid.
Conclusion
In the course of the experiment, it was shown that the changes in the parameters of AF are determined by an increase in its volume, an increase in inorganic (Na+, K+, Cl-, Pi, Ca2+) and organic (lactate, creatinine) components due to a decrease in reabsorption in the fetal kidneys.
6,1; 6,5; 7,1 0,027
tiles
References
1. Magann E.F., Sandlin A.T., Ounpraseuth S.T. Amniotic fluid and the clinical relevance of the sonographically estimated amniotic fluid volume: oligohydramnios. J. Ultrasound Med. 2011; 30(11): 1573-1585.
2. Beall M.H., van den Wijngaard J.P., van Gemert M.J., Ross M.G. Amniotic fluid water dynamics. Placenta. 2007; 28: 816-823.
3. Korenovsky Yu.V., Lytar I.A., Buryako-va S.I., Popovtseva A.V., Suzopov E.V., Obukhova L.E., Burkova T.V., Barsukova N.I., Remneva O.V., Fadeeva N.I. Amniotic fluid volume regulation. Obstetrics and gynecology. 2016; (2): 44-48.
4. Suzopov E.V., Lytar I.A., Popovtceva A.V., Korenovskii Y.V. Electrolytes concentration reference limits in amniotic fluid of rabbits on 27-28 day of gestation. Nephrology. 2017; 21(1):68-72.
5. Brace R.A., Cheung C.Y. Regulation of amniotic fluid volume: evolving concepts. Adv Exp Med Biol. 2014;814:49-68.
6. Thurlow R.W., Brace R.A.. Swallowing, urine flow and amniotic fluid volume responses to
prolonged hypoxia in the ovine fetus. Am J Obstet Gynecol. 2003; 189: 601-608.
7. Mann S.E., Nijland M.J., Ross M.G.. Ovine adaptations to chronically reduced urine flow: preservation of amniotic fluid volume. J Appl Physiol. 1996; 81: 2588-2594.
8. Manikandan K, Raghavan S. Amniotic fluid volume changes in response to furosemide induced maternal fluid shifts. J Pharmacol Pharmaco-ther. 2014; 5(2): 153-154.
9. Smirnov I.V., Bondarev A.A., Bryukhanov V.M., Postnikov P.S., Filimonov V.D. The mechanism of furosemide diuretic activity. The role of molecule hydrophobic segment. Siberian medical journal. 2011; 26(1-1): 123-126.
Contacts
Corresponding author: Shabalina Yulia Vadimovna, Senior Lecturer of the Department of General and Biological Chemistry, Clinical Laboratory Diagnostics of Altai State Medical University, Barnaul.
656038, Barnaul, Lenina Prospekt, 40.
Tel.: (3852) 566938.
Email: shabalinajv@gmail.com
Author information
Popovtseva Anna Valentinovna, Doctor of Clinical Laboratory Diagnostics in the Medical Laboratory, Diagnostic Center, OOO "SPF Helix", Saint-Petersburg.
190000, Saint-Petersburg, Naberezhnaya Reki Kar-povki, 5.
Tel. : (800)7000303.
Email: popovceva@gmail.com
Suzopov Egor Valeryevich, laboratory assistant of the Department of General and Biological Chemistry, Clinical Laboratory Diagnostics, Altai State Medical University, Barnaul. 656038, Barnaul, Lenina Prospekt, 40. Tel.: (3852) 241392. Email: suzopov1egor@gmail.com
Degtyareva Yulia Vladimirovna, Candidate of Biological Sciences, Associate Professor of the Department of General and Biological Chemistry, Clinical Laboratory Diagnostics, Altai State Medical University, Barnaul. 656038, Barnaul, Lenina Prospekt, 40. Tel.: (3852) 241392. Email: juliadegt@gmail.com
Zamyatina Svetlana Vladimirovna, Candidate of Medical Sciences, Associate Professor of the Department of General and Biological Chemistry, Clinical Laboratory Diagnostics, Altai State Medical University, Barnaul. 656038, Barnaul, Lenina Prospekt, 40. Tel.: (3852) 241392. Email: zamyatina_s_v@mail.ru
Bryukhanov Valery Mikhailovich, Doctor of Medical Sciences, Professor of the Department of Pharmacology, Altai State Medical University, Barnaul.
656038, Barnaul, Lenina Prospekt, 40. Tel.: (3852) 566812 E-mail: bvm@agmu.ru
Zharikov Aleksandr Yuryevich, Doctor of
Biological Sciences, Associate Professor, Head
of the Department of Pharmacology, Altai State
Medical University, Barnaul.
656056, Barnaul, ul. Papanintsev, 126.
Tel.: (3852) 241859.
E-mail: zharikov@agmu.ru
Korenovsky Yuri Vladimirovich, Candidate of Medical Sciences, Associate Professor, Head of the Department of General and Biological Chemistry, Clinical Laboratory Diagnostics, Altai State Medical University, Barnaul. 656038, Barnaul, ul. Papanitsev, 126. Tel.: (3852) 566938. E-mail: timidin@gmail.com
