Changes in Glycated Human Serum Albumin Binding Affinity for Losartan in the Presence of Fatty Acids In Vitro Spectroscopic Analysis

Conformational changes in human serum albumin due to numerous modifications that affect its stability and biological activity should be constantly monitored, especially in elderly patients and those suffering from chronic diseases (which include diabetes, obesity, and hypertension). The main goal of this study was to evaluate the effect of a mixture of fatty acids (FA) on the affinity of losartan (LOS, an angiotensin II receptor (AT1) blocker used in hypertension, a first-line treatment with coexisting diabetes) for glycated albumin—simulating the state of diabetes in the body. Individual fatty acid mixtures corresponded to the FA content in the physiological state and in various clinical states proceeding with increased concentrations of saturated (FAS) and unsaturated (FAUS) acids. Based on fluorescence studies, we conclude that LOS interacts with glycated human serum albumin (af)gHSA in the absence and in the presence of fatty acids ((af)gHSAphys, (af)gHSA4S, (af)gHSA8S, (af)gHSA4US, and (af)gHSA8US) and quenches the albumin fluorescence intensity via a static quenching mechanism. LOS not only binds to its specific binding sites in albumins but also non-specifically interacts with the hydrophobic fragments of its surface. Incorrect contents of fatty acids in the body affect the drug pharmacokinetics. A higher concentration of both FAS and FAUS acids in glycated albumin reduces the stability of the complex formed with losartan. The systematic study of FA and albumin interactions using an experimental model mimicking pathological conditions in the body may result in new tools for personalized pharmacotherapy.


Introduction
Human serum albumin (HSA), being the main protein in plasma, is essential in many processes taking part in the body. HSA performs key functions in maintaining homeostasis in the body, e.g., HSA controls the plasma oncotic pressure, modulates the fluid distribution between the body compartments, displays antioxidant and enzymatic properties, and inactivates toxic compounds [1][2][3][4]. HSA has the ability to transport many biologically active compounds through binding endo-and the exogenous compounds (e.g., fatty acids, metal ions, drugs, hormones, vitamins, toxins, and metabolites) [1,4].
One of the processes causing the loss of albumins original properties is the increased glycation in a state of hyperglycemia. Heterogeneous, stable compounds formed at the end of this process-Advanced Glycation End-Products (AGEs)-play a significant role in the development of chronic micro-and macroangiopathic diabetic complications as well as degenerative processes related to age [5,6].
Fatty Acids (FAs) perform many important function in living organisms, e.g., they are used as energy substrates in the β-oxidation process; as a building material for phospholipids, which are, in turn, used to create biological membranes; and they are precursors of important biological mediators, such as prostaglandins, leukotrienes, and thromboxanes [4,7].
FAs are also involved in intracellular transmission and take part in post-transcriptional modification processes [7]. As components of complex lipids, FAs play an important role in the electric and thermal isolation of the body, as well as provide it with mechanical protection. Due to the fatty acid low solubility in the blood plasma, albumin is the main transporter of FA [8]. The albumin-FA complex is in equilibrium with a very small fraction of unresolved FA dissolved in the plasma (less than 0.01% of the total pool).
Apart from the two main versatile ligand binding sites with a high affinity for diverse molecules referred to as 'Sudlow's sites'-site I (located in subdomain IIIA) and site II (in subdomain IIA) [9]-there are nine fatty acid binding sites in the HSA molecule (i.e., FA1-FA9), which are arranged in an asymmetrical manner and include all six subdomains [10] ( Figure 1). On one side, the non-polar bonds with fatty acids protect the tertiary structure of albumin against denaturation with guanidine hydrochloride, urea, and temperature. On the other side, there is a conformational change in the macromolecule and an increase in Cys-34 reactivity as a result of the exposure of the sulfhydryl residue [12]. Structural changes in Sudlow's site I concern the reorganization of hydrogen bonds between amino acid residues Tyr-150, Glu-153, Gln-196, His-242, Arg-257, and His-288, which leads to an increase in the area of the binding pocket and a polarity disorder.
In Sudlow's site II, there is a change in the conformation of the Leu-387 and Leu-453, and the breaking of Arg-348 and Glu-450 bonds. This allows the fatty acids, bound at FA3, to gain access to the polar region around FA4 [12]. Unmodified human serum albumin binds anions, when in combination with fatty acids, indicates an increased affinity for substances in the form of cations [13]. On one side, the non-polar bonds with fatty acids protect the tertiary structure of albumin against denaturation with guanidine hydrochloride, urea, and temperature. On the other side, there is a conformational change in the macromolecule and an increase in Cys-34 reactivity as a result of the exposure of the sulfhydryl residue [12]. Structural changes in Sudlow's site I concern the reorganization of hydrogen bonds between amino acid residues Tyr-150, Glu-153, Gln-196, His-242, Arg-257, and His-288, which leads to an increase in the area of the binding pocket and a polarity disorder.
In Sudlow's site II, there is a change in the conformation of the Leu-387 and Leu-453, and the breaking of Arg-348 and Glu-450 bonds. This allows the fatty acids, bound at FA3, to gain access to the polar region around FA4 [12]. Unmodified human serum albumin binds anions, when in combination with fatty acids, indicates an increased affinity for substances in the form of cations [13]. Many experiments have shown that the presence of fatty acids can have a significant impact on the process of drugs binding by albumin, particularly drugs with a high affinity for macromolecules. Fatty acids can compete for the HSA molecule binding sites or cooperate with drugs, wherein the FA affinity for albumin decreases with every filled macromolecule binding site [14,15].
It is important as ligands bound to one binding site can change the structure or number of other binding sites in the albumin molecule [12]. Due to the diversity present in the results concerning change in pharmacological action through research revolving around in vitro and in vivo studies of exogenous ligand interactions, the binding mechanisms of drugs in the presence of fatty acids with a transport protein requires thorough study.
The increasing occurrence of obesity related to, i.a., the overuse of saturated fatty acids in the diet is a predisposing factor to the appearance of metabolic syndrome, which significantly increases the risk of type 2 diabetes and cardiovascular disease in adults [16]. Its main components, apart from obesity, are primarily arterial hypertension, insulin resistance, and atherogenic dyslipidemia. Losartan (LOS, Figure 2) is one of the significant drugs used in the regulation of arterial hypertension as well as in the treatment for chronic heart failure and the prevention of cardiovascular diseases in order to reduce the risk of a stroke in patients with hypertension and left ventricular hypertrophy. As a selective and competitive, nonpeptide angiotensin II (AII) receptor antagonist, LOS blocks the vasoconstrictor and aldosterone-secreting effects of angiotensin II [17]. Many experiments have shown that the presence of fatty acids can have a significant impact on the process of drugs binding by albumin, particularly drugs with a high affinity for macromolecules. Fatty acids can compete for the HSA molecule binding sites or cooperate with drugs, wherein the FA affinity for albumin decreases with every filled macromolecule binding site [14,15].
It is important as ligands bound to one binding site can change the structure or number of other binding sites in the albumin molecule [12]. Due to the diversity present in the results concerning change in pharmacological action through research revolving around in vitro and in vivo studies of exogenous ligand interactions, the binding mechanisms of drugs in the presence of fatty acids with a transport protein requires thorough study.
The increasing occurrence of obesity related to, i.a., the overuse of saturated fatty acids in the diet is a predisposing factor to the appearance of metabolic syndrome, which significantly increases the risk of type 2 diabetes and cardiovascular disease in adults [16]. Its main components, apart from obesity, are primarily arterial hypertension, insulin resistance, and atherogenic dyslipidemia. Losartan (LOS, Figure 2) is one of the significant drugs used in the regulation of arterial hypertension as well as in the treatment for chronic heart failure and the prevention of cardiovascular diseases in order to reduce the risk of a stroke in patients with hypertension and left ventricular hypertrophy. As a selective and competitive, nonpeptide angiotensin II (AII) receptor antagonist, LOS blocks the vasoconstrictor and aldosterone-secreting effects of angiotensin II [17]. The aim of this study was to evaluate the effect of fatty acid (FA) mixtures with different saturated (PA-palmitic acid, MYR-myristic acid, and SA-stearic acid) and unsaturated (OA-oleic acid and LA-linoleic acid) fatty acids on the affinity of losartan for glycated human serum albumin-simulating the state of diabetes in the body. Individual fatty acid mixtures corresponded to the FA content in physiological state and in various clinical states proceeding with increased concentrations of saturated (FAS) and unsaturated (FAUS) acids. The binding properties of glycated albumin in the presence fatty acids and conformational changes of glycated human serum albumin were studied based on the quantitative analysis using absorption (UV-Vis) and fluorescence spectroscopy.
As has been well described in the literature, both UV-Vis and fluorescence spectroscopy, mainly the quenching of biomacromolecules fluorescence method, are very helpful in protein-ligand and protein-ligand-ligand interactions due to their high sensitivity, rapidity, and ease of implementation [18][19][20][21][22]. Fluorescence measurements can provide some information about the binding of small molecules to proteins, such as the binding mechanism, binding mode, binding constants, and binding sites, and are useful in drug development in the early stage of research [19].
The research regarding the influence of fatty acids on the structure and binding properties of glycated human albumin, which simulates the states of diabetes in the body, is important from the scientific point of view because the conformational transformation of the most important transport protein-serum albumin-due to the many modifications The aim of this study was to evaluate the effect of fatty acid (FA) mixtures with different saturated (PA-palmitic acid, MYR-myristic acid, and SA-stearic acid) and unsaturated (OA-oleic acid and LA-linoleic acid) fatty acids on the affinity of losartan for glycated human serum albumin-simulating the state of diabetes in the body. Individual fatty acid mixtures corresponded to the FA content in physiological state and in various clinical states proceeding with increased concentrations of saturated (FA S ) and unsaturated (FA US ) acids. The binding properties of glycated albumin in the presence fatty acids and conformational changes of glycated human serum albumin were studied based on the quantitative analysis using absorption (UV-Vis) and fluorescence spectroscopy.
As has been well described in the literature, both UV-Vis and fluorescence spectroscopy, mainly the quenching of biomacromolecules fluorescence method, are very helpful in protein-ligand and protein-ligand-ligand interactions due to their high sensitivity, rapidity, and ease of implementation [18][19][20][21][22]. Fluorescence measurements can provide some information about the binding of small molecules to proteins, such as the binding mechanism, binding mode, binding constants, and binding sites, and are useful in drug development in the early stage of research [19].
The research regarding the influence of fatty acids on the structure and binding properties of glycated human albumin, which simulates the states of diabetes in the body, is important from the scientific point of view because the conformational transformation of the most important transport protein-serum albumin-due to the many modifications that affect its stability and biological activity. This protein should be constantly monitored, especially in diseases and in the elderly. Monitoring the concentration of the drug-free fraction can help with optimizing pharmacotherapy as well as increase its effectiveness and avoid side effects.

Results and Discussion
2.1. The Interaction of Losartan with Glycated Human Serum Albumin in the Absence and in the Presence of Fatty Acids Based on the emission fluorescence spectra of glycated, defatted (af)gHSA and glycated in the presence of fatty acids (af)gHSA phys , (af)gHSA 4S , (af)gHSA 8S , (af)gHSA 4US , and (af)gHSA 8US albumin (5 × 10 −6 mol·L −1 ) (data not shown), an increase in the losartan (LOS) concentration (5 × 10 −6 mol·L −1 -5 × 10 −5 mol·L −1 ) in ligand-albumin systems causes a gradual decrease in the macromolecule fluorescence intensity. According to Stryer theory, the observed effect may be associated with the quenching fluorescence of excited fluorophores (tryptophanyl residue (Trp-214) and tyrosyl residues (Tyrs)) of glycated human albumins by the losartan molecule, which was found in no more than 10 nm proximity [18].
This distance makes it possible to transfer energy to the ligand molecule. In addition, in the LOS-(af)gHSA system (from a 0:1 to 10:1 molar ratio), after excitation at λ ex = 275 nm and λ ex = 295 nm, the shift in the defatted albumin fluorescence emission band towards shorter waves (blue shift) by 13 nm (∆λ max = 326-313 nm) and 2 nm (∆λ max = 337-335 nm) relative to the spectrum of the ligand-free albumin has been observed. The hypsochromic shift of maximum albumin fluorescence indicates the formation of a hydrophobic environment around the tryptophanyl (Trp-214) and residues tyrosyl (Tyrs) of (af)gHSA due to the interaction of LOS with albumin.
At the excitation λ ex = 295 nm, no shift has been recorded. Lakowicz explained that the emission of indole Trp-214 may be blue shifted if the group is buried within a native protein, and its emission may shift to longer wavelengths (red shift) when protein is unfolded [19]. Similarly, as in our previous work, the presence of acetohexamide (AH)-a drug with hypoglycemic activity and a sulfonylurea derivative of the first generation-also caused a blue shift of glycated human serum albumin in the absence of FA (af)gHSA spectra in AH-(af)gHSA [23].
Moeinpour et al., using a molecular dynamics simulation technique, also studied the interaction between losartan and glycated human serum albumin (gHSA). Based on the results visualized by Ligplus and Autodock ( Figure 11 from [25]), they concluded that LOS was located within the hydrophobic binding pocket of gHSA, and several phenyl groups of the drug interacted with the Glu-348, Glu-345, Val-346, Lys-373, Leu-369, Phe-349, Lys-364, Asp-372, Glu-360, and Asn-361 residues of subdomain IIB of gHSA through hydrophobic interaction.
Contrary to the study of LOS interaction with HSA, the specific hydrogen bonding interaction observed between the NH group of LOS and Asn-391 residue of albumin has not been identified. The environment of subdomain IIB, fatty acid high-(FA4), and low-(FA3, FA6, and FA7) affinity binding site, is likely the place where losartan can be located, and these sites could affect the binding.
Moreover, by the use of multiple spectroscopic methods, Moeinpour et al. also observed a blue shift of HSA maximum wavelength (fatty-acid-free human serum albumin), as well as its glycated form (gHSA) with an increasing amount of losartan [25]. This effect explained that the chromophore of HSA and gHSA was found to be directed towards more hydrophobic environments, and the conformation of the proteins was changed by the presence of the drug.
Fluorescence quenching curves present 5 × 10 −6 mol·L −1 glycated human serum albumin (af)gHSA in the absence and in the presence of fatty acids ((af)gHSA phys , (af)gHSA 4S , (af)gHSA 8S , (af)gHSA 4US , and (af)gHSA 8US ) fluorescence quotient in the absence (F 0 ) and in the presence of LOS (5 × 10 −6 mol·L −1 -5 × 10 −5 mol·L −1 ) (F) in the function of the drug:albumin molar ratio, λ ex = 275 nm and λ ex = 295 nm ( Figure 3). Contrary to the study of LOS interaction with HSA, the specific hydrogen bonding interaction observed between the NH group of LOS and Asn-391 residue of albumin has not been identified. The environment of subdomain IIB, fatty acid high-(FA4), and low-(FA3, FA6, and FA7) affinity binding site, is likely the place where losartan can be located, and these sites could affect the binding.
Moreover, by the use of multiple spectroscopic methods, Moeinpour et al. also observed a blue shift of HSA maximum wavelength (fatty-acid-free human serum albumin), as well as its glycated form (gHSA) with an increasing amount of losartan [25]. This effect explained that the chromophore of HSA and gHSA was found to be directed towards more hydrophobic environments, and the conformation of the proteins was changed by the presence of the drug.
Fluorescence quenching curves present 5 × 10 −6 mol•L −1 glycated human serum albumin (af)   The course of albumin fluorescence quenching curves illustrates the reduction in fluorescence intensity of human serum albumin (af)gHSA in the absence of fatty acids and with fatty acids ((af)gHSAphys, (af)gHSA4S, (af)gHSA8S, (af)gHSA4US, and (af)gHSA8US)) with the increase of losartan concentration in LOS-glycated albumin system (Figure 3a-c, in the main view and in the insert). The presence of fatty acids affects the ability of losartan to quench albumin fluorescence. Table 1 shows the percentage of fluorescence quenching (af)gHSA and (af)gHSAphys, (af)gHSA4S and (af)gHSA4US, (af)gHSA8S and (af)gHSA8US (5 × 10 −6 mol•L −1 ) for the highest concentration of LOS (5 × 10 −5 mol•L −1 ). The data collected in Table 1 show that the strongest quenching of albumin fluorescence in the presence of losartan with the increase of concentration was in the range of 59.34% and 77.80% for (af)gHSAphys at λex = 275 nm and λex = 295 nm, respectively.
This means that losartan is sufficiently close to protein tryptophanyl or/and tyrosyl residues (not more than 10 nm) and has the strongest affinity for (af) This demonstrates a higher losartan ability to absorb energy from excited fluorophores of albumin in the presence of fatty acids at physiological concentration ((af)gHSAphys) than from defatted albumin ((af)gHSA) and from albumin containing four times ((af)gHSA4S) and eight-times ((af)gHSA8S) higher amounts of saturated than unsaturated ((af)gHSA4US, (af)gHSA8US) fatty acids. This phenomenon is a result of conformational changes caused by the presence of fatty acids at physiological concentration or lower contents of saturated and unsaturated fatty acids.   Table 1 shows the percentage of fluorescence quenching (af)gHSA and (af)gHSA phys , (af)gHSA 4S and (af)gHSA 4US , (af)gHSA 8S and (af)gHSA 8US (5 × 10 −6 mol·L −1 ) for the highest concentration of LOS (5 × 10 −5 mol·L −1 ). The data collected in Table 1 show that the strongest quenching of albumin fluorescence in the presence of losartan with the increase of concentration was in the range of 59.34% and 77.80% for (af)gHSA phys at λ ex = 275 nm and λ ex = 295 nm, respectively. Table 1. Fluorescence quenching of LOS-(af)gHSA, LOS-(af)gHSA phys , LOS-(af)gHSA 4S , LOS-(af)gHSA 4US , LOS-(af)gHSA 8S , an LOS-(af)gHSA 8US systems and the fluorescence quenching percentage at λ ex = 275 nm and λ ex = 295 nm excitation wavelength; albumin and losartan concentrations were 5 × 10 −6 mol·L −1 and 5 × 10 −5 mol·L −1 , respectively. This means that losartan is sufficiently close to protein tryptophanyl or/and tyrosyl residues (not more than 10 nm) and has the strongest affinity for (af)gHSA phys molecule than for (af) wavelengths λ ex = 275 nm (in the main view) and λ ex = 295 nm (in the insert), has been observed.

Ligand-Albumin System
This demonstrates a higher losartan ability to absorb energy from excited fluorophores of albumin in the presence of fatty acids at physiological concentration ((af)gHSA phys ) than from defatted albumin ((af)gHSA) and from albumin containing four times ((af)gHSA 4S ) and eight-times ((af)gHSA 8S ) higher amounts of saturated than unsaturated ((af)gHSA 4US , (af)gHSA 8US ) fatty acids. This phenomenon is a result of conformational changes caused by the presence of fatty acids at physiological concentration or lower contents of saturated and unsaturated fatty acids.
As previously mentioned, after the excitation of albumin at λ ex = 295 nm, the observed emission of fluorescence comes almost exclusively from a tryptophanyl residue (Trp-214), while, for λ ex = 275 nm, this is from both Trp-214 and tyrosyl residues (Tyrs). The comparison between fluorescence quenching curves of glycated, defatted ((af)gHSA), and glycated in the presence of fatty acids ((af)gHSA phys , (af) (Table 1)), indicate the simultaneous participation of the Trp-214 residue located in subdomain IIA and Tyrs residues located in the IIA, IB, and IIB and subdomains in the interaction of LOS with albumin at the appropriate binding site. As reported in the literature, tyrosyl residues in position 401 (Tyr-401) and 411 (Tyr-411) located in the IIIA subdomain of albumin play a major role in drug binding [19,26]. The fluorescence quenching technique is not sufficient to indicate which Tyrs moieties are involved in LOS binding.
The mechanism of losartan interaction with albumin can be determined on the basis of Stern-Volmer curves (Equation (2)). Based on the data obtained from glycated, defatted (af)   On the other hand, static quenching leads to a reduction in fluorescence intensity when the ligand binds to the fluorophore molecule in its basic state (unexcited), reducing the population of excitable fluorophores [20]. The existence of dynamic and static quenching of human serum albumin fluorescence was obtained in our previous studies when the influence of piracetam (as a potential glycation inhibitor) on gliclazide-glycated albumin interaction was analyzed [27].
The linear F 0 /F = f [C LOS ] relationship for the other systems (Figure 4), indicates a dynamic or static mechanism of macromolecule fluorescence quenching in the environment of subdomains containing amino acid residues that are involved in the formation of the ligand-albumin complex. Moreover, the order of fluorescence quenching rate constants k q equals to 10 12 determined for LOS-glycated albumin system clearly indicates a static fluorescence quenching mechanism (Table 2), while according to Lakowicz, when the maximum value of the k q constant in the aqueous solution equals to 1 × 10 10 (mol −1 ·L·s −1 ), the dynamic fluorescence quenching mechanism occurs [19].    (3)) [21]. The plot of F 0 /∆F vs. 1/[C LOS ] is found to be linear with the intercept on the ordinate (Figure 5a,b). The reciprocal of the intercept gives the value of f a while the intercept/slope gives the value of the Stern-Volmer constants K SV . The obtained results have been collected in Table 2.  (3)) [21]. The plot of F0/∆F vs. 1/[CLOS] is found to be linear with the intercept on the ordinate (Figure 5a,b). The reciprocal of the intercept gives the value of fa while the intercept/slope gives the value of the Stern-Volmer constants KSV. The obtained results have been collected in Table 2. The Stern-Volmer constant is used to assess the availability of the quencher to the excited fluorophore. The growth of KSV value is associated with the increase of ligand molecule availability to the macromolecule and the formation of the complex in an excited state. As can be seen in the Table 2, the higher values of KSV constant obtained for the LOS-(af)gHSAphys system compared to KSV obtained for LOS-(af)gHSA indicate the location of losartan molecules closer to the fluorophores of glycated, fatted by fatty acids physiological mixture albumin ((af)gHSAphys) than glycated, defatted albumin (af)gHSA fluorophores.
The presence of fatty acids physiological mixture in glycated human serum albumin probably makes formation of LOS-(af)gHSAphys complex easier than the absence of fatty acids in the system (especially when the observed emission of fluorescence comes from both Trp-214 and tyrosyl residues (Tyrs)). The Stern-Volmer values and biomolecular quenching rate constants obtained for LOS-(af)gHSA4S and LOS-(af)gHSA8S are higher than KSV and kq values obtained for LOS-(af)gHSA4US and LOS-(af)gHSA8US (λex = 275 nm and λex = 295 nm).
Moreover, a two-fold increase in the amount of saturated fatty acids in the LOS-albumin system resulted in 23% decrease of KSV constant for λex = 275 nm and only 6% increase Ksv for λex = 295 nm. On the other hand, a two-fold increase in the amount of unsaturated fatty acids in the LOS-albumin system caused 38% and 63% decreases in KSV for λex = 275 nm and λex = 295 nm, respectively. These results indicate that LOS molecules locate at different distances to fluorophores of glycated albumin containing various amounts of saturated and unsaturated fatty acids. In addition, it can be seen that the availability of albumin (af)gHSAphys and (af)gHSA4S fluorophores (especially Trp-214 residues) for individual LOS binding sites is significantly facilitated ( Table 2).
To determine the nature of the interaction of losartan with glycated, defatted (af)gHSA and glycated in the presence of fatty acids albumin (af)gHSAphys, (af)gHSA4S, (af)gHSA8S, (af)gHSA4US, and (af)gHSA8US, binding isotherms were plotted in the LOS- The Stern-Volmer constant is used to assess the availability of the quencher to the excited fluorophore. The growth of K SV value is associated with the increase of ligand molecule availability to the macromolecule and the formation of the complex in an excited state. As can be seen in the Table 2, the higher values of K SV constant obtained for the LOS-(af)gHSA phys system compared to K SV obtained for LOS-(af)gHSA indicate the location of losartan molecules closer to the fluorophores of glycated, fatted by fatty acids physiological mixture albumin ((af)gHSA phys ) than glycated, defatted albumin (af)gHSA fluorophores.
The presence of fatty acids physiological mixture in glycated human serum albumin probably makes formation of LOS-(af)gHSA phys complex easier than the absence of fatty acids in the system (especially when the observed emission of fluorescence comes from both Trp-214 and tyrosyl residues (Tyrs)). The Stern-Volmer values and biomolecular quenching rate constants obtained for LOS-(af)gHSA 4S and LOS-(af)gHSA 8S are higher than K SV and k q values obtained for LOS-(af)gHSA 4US and LOS-(af)gHSA 8US (λ ex = 275 nm and λ ex = 295 nm).
Moreover, a two-fold increase in the amount of saturated fatty acids in the LOSalbumin system resulted in 23% decrease of K SV constant for λ ex = 275 nm and only 6% increase Ksv for λ ex = 295 nm. On the other hand, a two-fold increase in the amount of unsaturated fatty acids in the LOS-albumin system caused 38% and 63% decreases in K SV for λ ex = 275 nm and λ ex = 295 nm, respectively. These results indicate that LOS molecules locate at different distances to fluorophores of glycated albumin containing various amounts of saturated and unsaturated fatty acids. In addition, it can be seen that the availability of albumin (af)gHSA phys and (af)gHSA 4S fluorophores (especially Trp-214 residues) for individual LOS binding sites is significantly facilitated ( Table 2).
To determine the nature of the interaction of losartan with glycated, defatted (af)gHSA and glycated in the presence of fatty acids albumin (af)gHSA phys , (af)gHSA 4S , (af)gHSA 8S , (af)gHSA 4US , and (af)gHSA 8US , binding isotherms were plotted in the LOS-(af)gHSA and LOS-(af)gHSA phys (Figure 6a Similarly, as in our previous paper [28], where the interaction of tolbutamide and losartan with human serum albumin in hyperglycemia states were studied, a non-linear relationship r = f ([L f ]) was observed ( Figure 6). The non-linear shape of the binding isotherms obtained for LOS-(af)gHSA and LOS-(af)gHSA phys (Figure 6a Similarly, as in our previous paper [28], where the interaction of tolbutamide and losartan with human serum albumin in hyperglycemia states were studied, a non-linear relationship r = f([Lf]) was observed ( Figure 6). The non-linear shape of the binding isotherms obtained for LOS-(af)gHSA and LOS-(af)gHSAphys (Figure 6a This means that losartan binds not only to its specific binding sites in glycated, defatted and in the presence of fatty acids albumin but also non-specifically interacts with the hydrophobic fragments of its surface [29]. However, the shape of the binding isotherms for glycated human serum albumin with fatty acids containing eight-times more unsaturated fatty acids in relation to the physiological value, indicates the occurrence of only the non-specific nature of losartan binding to (af)gHSA8US (Figure 6c).
Specific binding is characterized by high affinity and low binding capacity, while non-specific binding is characterized by low affinity and unlimited drug binding capacity This means that losartan binds not only to its specific binding sites in glycated, defatted and in the presence of fatty acids albumin but also non-specifically interacts with the hydrophobic fragments of its surface [29]. However, the shape of the binding isotherms for glycated human serum albumin with fatty acids containing eight-times more unsaturated fatty acids in relation to the physiological value, indicates the occurrence of only the non-specific nature of losartan binding to (af)gHSA 8US (Figure 6c). Specific binding is characterized by high affinity and low binding capacity, while nonspecific binding is characterized by low affinity and unlimited drug binding capacity [29]. Regardless of the course of binding isotherms (Figure 6), the losartan-glycated albumin interaction is likely characterized by a specific type of binding because, in physiological environments, the drug:albumin molar ratio is much smaller than 1:1 and equals to 1:500.
There are many methods for the calculation of association constant (K a ) that characterizes the stability of formed drug-albumin complex for the determination the number of drug molecules (n) associated with one albumin molecule at equilibrium, or for the prediction an existence of one or more independent classes of binding sites. In the present work, specific binding of losartan to glycated human serum albumin in LOS- (af) In the Scatchard equation, the concentration of the bound ligand to the protein is the independent variable, while, in the Klotz equation, the independent variable is the reciprocal of the free ligand fraction. To study the possible cooperation of losartan binding to the macromolecule, the Hill interaction factors (nH) were determined by the use of the Hill equation (the dependence of log[r/(1 − r)] on log[Lf], Equation (6), Figure 9). The number of losartan molecules (n) forming a complex with one molecule of (af)gHSA, (af)gHSAphys, (af)gHSA4S, (af)gHSA4US, (af)gHSA8S, and (af)gHSA8US at equilibrium state for a specific class of binding sites was also obtained. The binding parameters (Ka, n) and Hill nH coefficient (interaction factor) are summarized in Table 3.   In the Scatchard equation, the concentration of the bound ligand to the protein is the independent variable, while, in the Klotz equation, the independent variable is the reciprocal of the free ligand fraction. To study the possible cooperation of losartan binding to the macromolecule, the Hill interaction factors (n H ) were determined by the use of the Hill equation (the dependence of log[r/(1 − r)] on log[L f ], Equation (6), Figure 9). The number of losartan molecules (n) forming a complex with one molecule of (af)gHSA, (af)gHSA phys , (af)gHSA 4S , (af)gHSA 4US , (af)gHSA 8S , and (af)gHSA 8US at equilibrium state for a specific class of binding sites was also obtained. The binding parameters (K a , n) and Hill n H coefficient (interaction factor) are summarized in Table 3.  Table 3. Association constants K a (mol −1 ·L), mean number of LOS moles bound with one mole of (af)gHSA, (af)gHSA phys , (af)gHSA 4S , (af)gHSA 4US , (af)gHSA 8S , and (af)gHSA 8US (n), the Hill coefficient (n H ) in The LOS-albumin systems; λ ex = 275 nm, λ ex = 295 nm.
The association constants K a determined from the Scatchard and the Klotz relationships for the complexes LOS-(af)gHSA 8US for λ ex = 275 nm and λ ex = 295 nm prove the specific nature of losartan binding within the albumin (Table 3). For LOS-(af)gHSA complex, the association constants K a are the same (for λ ex = 275 nm) and not much lower (for λ ex = 295 nm) than the constants K a values obtained for losartan-(af)gHSA phys complex (Table 3), which indicates that LOS has the same affinity for (af)gHSA and (af)gHSA phys albumin binding sites.
For the LOS-albumin complex with a two-times greater amount of saturated ((af)gHSA 8S ) and unsaturated ((af)gHSA 8US ) fatty acids, the K a constants are smaller than those obtained for the LOS-(af)gHSA 4S and LOS-(af)gHSA 4US for λ ex = 275 nm and 295 nm (Table 3). This means that a higher concentration of both saturated and unsaturated fatty acids in glycated albumin reduces the stability of the complex formed with losartan. For LOS-(af)gHSA, LOS-(af)gHSA phys , LOS-(af)gHSA 4S , LOS-(af)gHSA 8S , LOS-(af)gHSA 4US , and LOS-(af)gHSA 8US complexes, an average of one ligand molecule binds to one albumin molecule (n ≈ 1).
The Hill interaction coefficient n H equals unity (n H ≈ 1) and indicates a lack of cooperativity in the binding of LOS to albumins in the vicinity of Trp-214 and Tyrs residues. This is the same value of n H that we obtained in our previous work [23] when we determined the Hill interaction coefficient for acetohexamide-albumin complex with four-and eightfold higher unsaturated and eight-fold higher saturated fatty acids amount compared to physiological value.
Based on the in vitro results, the fatty acids affect losartan binding to glycated human serum albumin. It can be assumed that, under conditions of abnormal fat content in the body, the pharmacokinetics of the drug may be disturbed. It is noteworthy that during a treatment with losartan it is important to control the amount of fatty acids supplied to the body with diet and/or in the form of supplements. Stronger binding of LOS to albumin weakens its therapeutic effect; however, on the other hand, the free drug fraction has potentially toxic side effects that can be dangerous to the patient's health. The research suggests the need for individual dose selection, especially for the obese patients with chronic diseases.

Structural Modification of Glycated Human Serum Albumin Caused by Fatty Acids
The physicochemical and biological properties of proteins are directly dependent on their spatial structure. It is for this reason that studies that allow us to observe conformational changes in protein caused by various modifications are important. A number of structural modifications, especially in the tertiary confirmation of human serum albumin, are attributed to, e.g., protein glycation [30].
In this part of the study, we examined whether fatty acids cause additional conformational changes in the tertiary structure of glycated albumin, which simulates diabetes in the body. It is of key importance in planning therapy because the strength and nature of the drug's interactions with its main distributor may change in the presence of coexisting diabetes and obesity (Section 2.1).
Although circular dichroism (CD) plays an important role in the study of protein folding as it allows the characterization of secondary and tertiary structure of proteins in native, unfolded and partially folded states [31], the CD spectra of the studied proteins were impossible to register due to the presence of the introduced fatty acids. Hence, to indicate changes in the tertiary structure of glycated albumin induced by the presence of fatty acids, fluorescence spectroscopy was used.
For this purpose, we compared the emission and synchronous fluorescence spectra of glycated human serum albumin ((af) show the conformational changes in the environment of the tryptophanyl and tyrosine residues of glycated human serum albumin influenced by fatty acids. It is well known that the wavelength of 275 nm excites not only Trp-214 but also tyrosine residues and it is impossible to separately observe the fluorescence of these fluorophores. Synchronous fluorescence spectroscopy allows for separation of the emission spectra originating from the Trp-214 and Tyrs (as illustrated in Figures 10, 11 and 12b, main view), which results more specific information about the structure of the macromolecules. According to literature data [25,28,32], the synchronous fluorescence spectra were obtained considering the wavelength intervals Δλ = 60 nm and Δλ = 15 nm to evidence the Trp-214 and Tyrs, respectively (Δλ = λem − λex).       Synchronous fluorescence spectroscopy allows for separation of the emission spectra originating from the Trp-214 and Tyrs (as illustrated in Figures 10, 11 and 12b, main view), which results more specific information about the structure of the macromolecules. According to literature data [25,28,32], the synchronous fluorescence spectra were obtained considering the wavelength intervals ∆λ = 60 nm and ∆λ = 15 nm to evidence the Trp-214 and Tyrs, respectively (∆λ = λ em − λ ex ). The fluorescence of human serum albumin fluorophores is sensitive to the changes of HSA tertiary structure and environmental properties. Slight structural changes in albumin near the Trp-214 and Tyrs residues affect the fluorescence intensity (Fmax) and position of maximum fluorescence (λmax) [33]. A blue shift of λmax indicates that the Trp-214 and Tyrs residues are located in a more hydrophobic environment, while a red-shift of λmax implies that the amino acid residues are in a polar environment and are more exposed to the solvent [34].
Using Δλ = 15 nm (Figures 10-12a, main view) and Δλ = 60 nm (Figures 10-12b, main view) no changes were observed in the maximum emission wavelength of (af)gHSA and (af)gHSAphys ( Figure 10   The fluorescence of human serum albumin fluorophores is sensitive to the changes of HSA tertiary structure and environmental properties. Slight structural changes in albumin near the Trp-214 and Tyrs residues affect the fluorescence intensity (F max ) and position of maximum fluorescence (λ max ) [33]. A blue shift of λ max indicates that the Trp-214 and Tyrs residues are located in a more hydrophobic environment, while a red-shift of λ max implies that the amino acid residues are in a polar environment and are more exposed to the solvent [34].
The fluorescence intensity of both types of fluorophores in the (af)gHSA spectrum is lower than in the (af)gHSA phys , (af)gHSA 4S , (af)gHSA 8S , (af)gHSA 4US , and (af)gHSA 8US spectra (Table 4) Red Edge Excitation Shift (REES) is an another method to directly monitor of the region surrounding the tryptophanyl residue of glycated, deffated, and glycated in the presence of fatty acids human serum albumin [33,36]. In order to study the REES effect, fluorescence spectra of glycated human serum albumin in the absence (af)gHSA and in the presence of fatty acids (af)gHSA phys , (af)gHSA 4S , (af)gHSA 8S , (af)gHSA 4US , and (af)gHSA 8US excited at λ ex = 290 nm, λ ex = 295 nm, and λ ex = 300 nm wavelengths were recorded (data not shown).
Emission fluorescence spectra of (af)gHSA Trp-214 residue are different than for Trp-214 of (af)gHSA phys , (af)gHSA 4S , (af)gHSA 8S , (af)gHSA 4US , and (af)gHSA 8US at all excitation wavelengths. A slight red-shift maximum emission fluorescence of (af)gHSA phys (∆λ em = 2 nm), The degree of macromolecule fluorescence quenching by the LOS was determined relative to the fluorescence of the non-ligand albumin solutions. Due to the inner filter effect (IFE) caused by the presence of the drug, the recorded fluorescence was corrected using the following formula (Equation (1)) [19]: where F cor and F obs are the corrected and observed fluorescence intensity, respectively; A ex and A em are the absorbance at excitation (λ ex = 275 nm or λ ex = 295 nm) and emission wavelength for (af)gHSA, (af)gHSA phys , (af)gHSA 4S , (af)gHSA 8S , (af)gHSA 4US , and (af)gHSA 8US , respectively.

Analysis of Fluorescence Spectra-Calculation of the Stern-Volmer and Association Constants
Based on the calculated fluorescence emission intensities in the absence and in the presence of fatty acids glycated human serum albumin, curves of (af)gHSA, (af)gHSA phys , (af)gHSA 4S , (af)gHSA 8S , (af)gHSA 4US , and (af)gHSA 8US fluorescence quenching by losartan (LOS) ( F F 0 vs. ligand:albumin molar ratio, where F and F 0 is the fluorescence intensity at the maximum wavelength of albumin in the presence and absence of a quencher, respectively) were drawn.
The quenching effect (static and/or dynamic) fluorescence of (af)gHSA, (af)gHSA phys , (af)gHSA 4S , (af)gHSA 8S , (af)gHSA 4US , and (af)gHSA 8US , the Stern-Volmer constants K SV , the bimolecular quenching rate constants k q (k q = K SV /τ 0 ), and maximum available fluorescence fraction of all albumin f a fluorophores were analyzed on the basis of the Stern-Volmer equation (Equation (2)) [20]: where k q is the bimolecular quenching rate constant [mol −1 ·L·s −1 ]; τ 0 is the average fluorescence lifetime of albumin without of quencher τ 0 = 6.0 × 10 − The quenching parameters (K SV , k q ) and f a for a system with non-linear Stern-Volmer relationship were calculated using the Stern-Volmer equation modified by Lehrer (Equation (3)) [21]: where f a is the fractional maximum protein fluorescence accessible for the quencher.  [29]. From the Scatchard (Equation (4)) [37] and the Klotz (Equation (5)) [38] curves, the values of K a association constants and n the number of binding sites in the albumin molecule were determined. r