Startseite Fluorescence biomarkers of malignant melanoma detectable in urine
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Fluorescence biomarkers of malignant melanoma detectable in urine

  • Ivana Špaková EMAIL logo , Katarína Dubayová , Vladimíra Nagyová und Mária Mareková
Veröffentlicht/Copyright: 7. August 2020
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Open Chemistry
Aus der Zeitschrift Open Chemistry Band 18 Heft 1

Abstract

Malignant melanoma (MM) is a cancerous transformation of melanocytes. It is a disease with the worst response to therapy and, compared to other malignancies, presents much earlier with metastases. MM still belongs to relatively late-detected malignant diseases. Even so, the MM mortality rate is up to 96% for a relatively small incidence (5%). The gold standard for MM diagnosis is a histopathological examination that requires invasive surgery. An invasive sampling method of a biological material can be a stressful factor for the patient, which is often the reason why patients do not seek medical assistance as soon as possible. Our goal was to find a link between metabolites in urine and the stage of MM. Two excitation peaks at 360–370 nm and 450 nm were characterised in spectra of urine samples. The emission spectra have shown one significant peak at 410–460 nm. After addition of glutathione reductase to the samples, fluorescence dropped down only in patient samples and hidden fluorophores appeared. Malignant diseases are associated with the presence of specific metabolites that can be detected fluorescently in biological material such as urine, which can be a suitable alternative for an early detection of cancer or for tracking changes during and after treatment.

Keywords: NAD(P)H; lipofuscin; HIF-1α; MITF-M; IGF1

1 Introduction

Urine contains a number of endogenous fluorophores that can be used to monitor the course of a disease, the response to treatment or identify the potential threat of disease development. Known compounds with altered fluorescence intensity observed in case of some cancers (bladder, breast, etc.) include free (unbound) NAD(P)H molecules, flavins, porphyrins, proteins and pigments [1,2,3,4].

NADH and NADPH (collectively referred to as NADH hereafter) are increasingly produced by cancer cells as a consequence of their preference for cytosolic metabolism, the so-called Warburg effect [5]. Another feature of neoplastic tissue is the accumulation of porphyrins, the so-called porphyria [6,7]. Water-soluble porphyrins (5-aminolevulinic acid [ALA], porphobilinogen [PBG], uroporphyrin) are partially excreted into the urine and their amount increases with disease progression.

The tumour microenvironment is also defined by an extracellular matrix rearrangement. The physiological presence of protein structures (collagens, elastin, laminins) is reduced by their degradation. Elastin fibres surround the nevus root [8]. Melanomas have significantly fewer elastic fibres in the melanocyte roots. Melanoma cells exert pressure on the elastin layer in the papillary dermis, forming a compressed layer that distinguishes melanoma from the nevus [8]. The elastin layer is still present, however less evident. Elastin fragments affect the presence of matrix metalloproteinases (MMP-2, -4) and activate melanoma cells [9]. Higher levels of elastin remnants in tumours are associated with a higher stage of disease and metastasis [10]. The breakdown of elastin and its loss increases with Breslow tumour categorisation, mitotic degree as well as tumorigenic transcriptional and growth factors aberration, which are negative factors for malignant melanoma (MM) progression.

Another endogenous fluorophore, the concentration of which increases as a result of the presence of melanocyte cancer transformation, is lipofuscin (water-soluble pigment) [11]. Accumulation of aberrant lipofuscin results in a marked pigmentation of the affected tissue [12], which usually appears as an abnormally brown to black colouration. Lipofuscin accumulation has been reported in retinal pigment epithelium cells and macrophages in several types of benign tumours, lesions simulating tumours and MM and choroid tumours [13]. Choroid tumours, unlike MM, do not exhibit typical tumour pigmentation; they are almost invisible ophthalmoscopically, but the presence of lipofuscin can be detected by fluorescence. Lipofuscin is produced as a waste material of intracellular structures accumulated in the lysosomes of affected cells [14]. The formation of the lipoid pigment is the result of decreased cysteine protease activity in the lysosomes, which diminishes by age, and it is therefore also referred as the old-age pigment [15,16]. Its accumulation under conditions of oxidative stress due to reactive oxygen species (ROS) attack on lysosomes and mitochondria (mitochondria undergo autophagocytosis) leads to cell damage by destabilising membrane structures through the release of lipofuscin [17,18]. Non-physiological pigment accumulation (not associated to high age) is a sign of the tumour transformation of melanocytes. Implication of degenerative changes due to the development of MM, lipofuscin may occur in the urine [19].

The accumulation of metabolites, proteins or pigments is narrowly linked to the metabolic state of the cells which is genetically regulated. Malignant tumours are usually characterised by an insufficient supply of nutrients and oxygen to all areas of the tumour, resulting in so-called hypoxic or pseudohypoxic regions [20]. Cells in these malignant structures increasingly express hypoxia-inducible factor 1 alpha (HIF-1α) which induces the expression of such regulatory genes that promote the adaptation of the cell to reduced oxygen and nutrients [21]. Activation of HIF-1α in an effort to minimise the negative effects of low oxygen reduces cell demand for oxygen by responding to oxidative phosphorylation (OXPHOS) which as one of the main processes requires the most oxygen [22]. This adaptation is primarily mediated by favouring glycolysis as the main energy metabolic process [23]. Disconnection of OXPHOS is the cornerstone for the formation of ROS (ROS back-stabilises HIF-1α in hypoxia as well as normoxia) as a result of reduced respiratory complex activity [24]. Detachment of OXPHOS is a reversible process that when returned to the physiological state of oxygen leads to reduced oxygen consumption in response to activated HIF-1α [25]. This dynamic equilibrium depends on the ability of oxygen to cross the membrane, which is affected by cholesterol, and lipid metabolism (closely linked to the viability of mitochondria and OXPHOS) [21]. Dysregulation of OXPHOS by HIF-1α hyperactivation leads to limitation of pyruvate entry into the citrate cycle to mitigate the damage caused by non-physiological ROS generation [26]. Therefore, non-glucose metabolites especially from glutamine metabolism are favoured in the citrate cycle [27,28]. Originated by NADH, metabolic processes in mitochondrial matrix and cytosol do not regenerate on respiratory complexes but are transferred by the malate-aspartate shuttle system to the cytosol, where they accumulate and participate in the Warburg effect [29]. Abnormal stabilisation of HIF-1α and NADH leads to malignant transformation and poor prognosis [30].

The mitochondrial protector is an insulin-like growth factor polypeptide [31,32] which is involved in the regulation of mitochondrial function, oxidative stress, and normal cell growth. Aberrant expression of IGF-1 increases expression of genes and proteins as BCL-2 and BCL-X(L) [33,34] which leads cells to immortality by resistance to apoptotic protective stimuli. Microphthalmia-associated transcription factor-melanocyte specific isoform (MITF-M) is involved in the maintenance of active mitochondrial metabolism [35], whose dysregulation leads to inhibition of OXPHOS. HIF-1α decreases the expression of MITF-M [36] and increases the invasivity of the MM phenotype [37]. The main role of the MITF pathway is to regulate the cellular response to ROS stress by inducing tyrosinase synthesis which is a rate-limiting enzyme for melanin synthesis [38]. MITF-M depletion attenuates melanocyte response to autophagy induced by insufficient nutrient and oxygen intake (starvation) [39]. Downregulation of MITF leads to accumulation of lipofuscin [40,41] due to degradation of melanin.

Since NADH, FAD and lipofuscin belong to the endogenous fluorescent parameters which significantly change upon the transformation of melanocytes into malignant tumour, we explored their diagnostic potential in monitoring of MM progression.

2 Material and methods

2.1 Biological material

We studied the fluorescence intensity of endogenous fluorophores present in the urine and gene expression of genes related to starvation under hypoxia of MM patients and healthy probands. The demographic description of experimental groups is shown in Table 1.

Table 1

The demographic description of experimental groups

ProbandsAverage age
MM patients 4651
Controls 1044
SSM1148
ALM248
NM850
LM259
MMIS250
Undefined854
pT1ab850
pT2ab552
pT3ab849
pT4ab1251
Undefined1354

2.2 Sampling and analysis

The urine samples were centrifuged for 5 min under 2,000 rpm at room temperature. Samples were diluted with deionised water in a 1:25 ratio. Excitation-emission matrices (EEMs), excitation (EX), absorption (AB) and specific emission fluorescence spectra (EM) were measured using a Horiba Dual FL dual spectrophotometer in the range 300–510 nm for the absorption spectra, in the range of 350–730 nm for EM (λEX = 340 nm), and in the excitation range of 240–800 nm (λEM = 536 nm). Addition of 2 µL of glutathione reductase (GR) to 2 mL of urine samples and further analysis of emission spectra and EEM spectra were performed. GR is a homodimer containing FAD per monomer that restores intracellular glutathione (GSH) by reducing glutathione disulphide (GSSG) in the presence of NADH or NADPH (as hydrogen donor) [42].

The expression levels of the HIF-1α, IGF-1 and MITF-M genes were analysed from blood (mRNA isolation by QIAamp RNA Blood Mini Kit (50), Cat. no.: 52304, Qiagen), reverse RT-PCR was performed by ProtoScript First Strand cDNA Synthesis Kit (E6300S, New England BioLabs) and quantification of the gene product was done by qRT-PCR (Maxima Hot Start PCR Master Mix (2x), K1059, Fermentas Life Sciences) and by Rotor-Gene Q (Ser. No.: R1114130, Qiagen).

2.3 Statistics

The obtained data were analysed with OriginPro 8 SR2 v8.0891 (OriginLab Corporation) and GraphPad Prism 5.04 (GraphPad Software). The presented data are mean ± standard error of the mean in three independent experimental repeats. The unpaired t-test, F-test of variances and multiple t-test were used to compare the data obtained in the two groups. When comparing values among multiple groups, the one-way and two-way ANOVA statistical methods were used to evaluate statistical significance. Statistically significant results were found to have a p value (level of significance) below 0.05.

  1. Informed consent: Informed consent has been obtained from all individuals included in this study.

  2. Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance with the tenets of the Helsinki Declaration, and has been approved by the authors’ institutional review board or equivalent committee. Permission from the ethics committee was given by Ethics commission of Pavol Jozef Šafárik University in Košice, Faculty of Medicine is 20N/2016. This work was a part of doctoral thesis.

3 Results

Using EEM, excitation spectra and emission spectra, we analysed urine samples from patients with MM and healthy probands. We focused on the excitation spectra in the range of 300–500 nm, where two maxima were found; first found within the range 340–360 nm can be attributed mainly to NADH, collagens and elastin and second at 450 nm associated with porphyrins (Figure 1a). Typically, the maximum at 450 nm was more pronounced in MM patients. Based on the measured data, we have produced graphical representations of mean intensities for a maximum of 365 nm and at 450 nm in patients and in the controls (Figure 1b, c and Table 2). The calculated REX = 450/365 ratio for patient samples and for the controls is shown in Figure 1d and Table 2.

Figure 1 Excitation spectra (a), levels of excitation maxima at 365 (b) and 450 nm (c), ratio of excitation maxima 450/365 nm (d), absorption spectra (e), absorbance maxima at 280/340/375/450 nm (f).
Figure 1

Excitation spectra (a), levels of excitation maxima at 365 (b) and 450 nm (c), ratio of excitation maxima 450/365 nm (d), absorption spectra (e), absorbance maxima at 280/340/375/450 nm (f).

Table 2

Intensity values for absorption, excitation and emission spectra (significance, p > 0.05 is non-significant (ns); p < 0.05 is *; p < 0.01 is **; p < 0.001 is ***)

MM patientsControlsSignificance
Average (µA)±SDAverage (µA)±SD
λEX = 365 nm3010.94635.432844.00671.74ns
λEX = 450 nm2750.26656.241990.6010.10ns
REX = 450/3650.890.230.740.18ns
λAB = 280 nm1.040.471.490.11***
λAB = 340 nm0.450.150.240.07ns
λAB = 375 nm0.420.110.210.08ns
λAB = 450 nm0.370.090.210.08ns
λEM = 410 nm (GR)3528.452938.0411711.641512.40
λEM = 440 nm8287.582356.4211140.03863.98ns
λEM = 440 nm (GR)3462.851028.901073.781048.09***
λEM = 460 nm6423.261656.429905.101032.54**
λEM = 460 nm (GR)3112.79775.318505.651394.84***
λEM = 525 nm2356.13851.201180.24227.54***
λEM = 525 nm (GR)2702.53256.082573.22334.41***
λEM = 565 nm1353.48492.071376.66103.66ns
λEM = 565 nm (GR)480.8643.00801.5033.49***
λEM = 620 nm441.15105.43387.9713.95ns
λEM = 620 nm (GR)370.14102.40391.0213.72ns
REM = 620/4600.070.040.050.01*
REM = 620/460 (GR)0.180.130.040.01***

Absorption spectra were characterised by 1.42 times decrease in maxima at 280 nm which represent total value of proteins and amino acids (average of absorbance patients in Table 2). Next, the spectra showed increase at λAB = 340 nm, λAB = 375 nm and λAB = 450 nm in patient samples (Figure 1e, f and Table 2). Using two-way ANOVA, the Sidak’s multiple comparison test showed strong significant decrease at λAB = 280 nm at p < 0.0001 and non-significant increase at λAB = 340, 375 and 450 nm.

Furthermore, we focused on emission spectra in the studied group in the range of 350–730 nm after excitation at λEX = 340 nm. Emission spectra were characterised by one single peak in the range of 410–460 nm which consists of multiple fluorescence peaks for NADH, elastin/collagen, FAD, etc. Patient samples were generally characterised by a higher fluorescence intensity of this maximum than the control samples (Figure 2a). The difference before and after addition of GR in the range 355–500 nm with the maxima at 440 nm or 460 nm (possibly NADH and NADPH emission after excitation by 340 nm) is caused by NADPH fluorescence intensity which is sensitive to dehydrogenation by GR [43]. Upon the addition of GR to the urine, the emission of free NADH dropped while peaks representative mainly for elastin/collagen (410 nm), NADH (440, 460 nm), flavins (FAD 525 nm), porphyrins (basic 585 nm, acidic/neutral 620–630 nm) and pigments (lipofuscin soluble in water 565 nm) showed up (Figure 2b). Samples with GR were characterised by a significant reduction in fluorescence within the 410–460 nm region in MM patients, while no significant effect was observed in the controls. After the removal of NADH emission using GR, some maxima at 410, 440, 460, 525 and 565 nm were discovered (Figure 2c–f). Other peaks that were visible after the addition of GR were expected for NADH at 440 nm, expected for NADPH at 460 nm, presupposed for FAD at 525 nm, assumed for lipofuscin-like pigments at 565 nm and presumed for acid/neutral porphyrins at 620 nm (values of intensities in Table 2). The REM = 620/460 ratio and the REM = 620/460 ratio after addition of GR for patient samples and for controls are in Table 2. Using comparison multiple t-test two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli between samples and controls gives significant result with p = 0.031 between patients and controls, and significance with p < 0.001 between patients and controls after addition of GR (multiple t-test two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli is a procedure for controlling the false discovery rate of a family of hypothesis tests where the null hypothesis is true).

Figure 2 Emission spectra of urine samples ± GR (a), detail of emission region 350–750 nm (b), emission maxima (±GR) at 440 nm (c), 460 nm (d), 565 nm (e), 620 nm (f).
Figure 2

Emission spectra of urine samples ± GR (a), detail of emission region 350–750 nm (b), emission maxima (±GR) at 440 nm (c), 460 nm (d), 565 nm (e), 620 nm (f).

From the intensity difference before and after addition of GR at the range 355–500 nm, we calculated the capacity of area under the curve and percentage presume of NAD(P)H, which was 96% in average for the patients compared to average of the area for patients and 51% in average for the patients compared to average of the area for controls (Figure 3).

Figure 3 Amount area under the plot probable of NADH in urine samples based on emission spectra.
Figure 3

Amount area under the plot probable of NADH in urine samples based on emission spectra.

After distribution of measured excitation and emission maxima after addition of GR by type of MM (Superficial spreading melanoma [SSM], nodular melanoma [NM], acral lentiginous melanoma [ALM], lentigo melanoma [LM], malignant melanoma in situ [MMIS]), we can see the tendency that there is dependency on emission maxima at 440 and 460 nm which increases with increasing malignancy (increasing pT stage of MM). On the contrary, after addition of GR we can see the decrease of the fluorescence intensity at 440 and 460 nm which corresponds with incorporation of NADH and NADPH into enzyme (Figure 4a and b). For ulcerated and non-ulcerated tumours (Figure 4c), we determined opposite leaning that ulcerated tumours had slightly lower fluorescence than non-ulcerated tumours. Based on distribution of Melan A (Figure 4d), we did not determine any significant change between MM patients. The statistics of unpaired t-test, F-test of variances (no association among groups analysis) and multiple t-test of emission intensities are in supplementary data. The two-way ANOVA showed strong significant change with p value <0.0001 if compare differences between types of MM at λEM = 440, 460, and 525 nm by the Dunnett’s multiple comparisons test (Dunnett’s test is pairwise comparison of multiple treatment groups with a single control group). At λEM = 565 nm, the significance difference was between LM vs controls at the p level 0.0075 and undefined type of MM vs controls at the p level 0.013. The rest were non-significant (see supplementary data). Next, the two-way ANOVA gave the strong significant change with the p level <0.0001 if compare differences between types of MM at λEM = 440, 460, 525 nm (except for pT4ab vs controls which was at the level 0.013) and 565 nm by the Dunnett’s multiple comparisons test. At λEM = 620 nm, there was no significance between groups (see supplementary data). Finally, the two-way ANOVA showed up the strong significant change at the p level <0.0001 if compare differences between presented and non-presented melan A parameters at λEM = 440 and 460 nm by the Dunnett’s multiple comparisons test. At λEM = 525 nm, the significance difference was between presented melan A vs controls with p = 0.008, non-presented melan A vs controls with p = 0.029, undefined vs controls with p = 0.008, and at 620 nm there was no significance between groups (see supplementary data). When comparing emission fluorescence intensities at 565 nm (presumably lipofuscin), it shows the significant increase in the intensity with increasing the pT stage of MM after addition of GR (Figure 5, supplementary data).

Figure 4 Fluorescence intensities for excitation at 365 and 450 nm, and emission at 410/460/565 and 620 nm (±GR) based on EEM by MM type (a and b), MM pT stage (c and d), MM ulceration (e and f), MM melan A (f and g).
Figure 4

Fluorescence intensities for excitation at 365 and 450 nm, and emission at 410/460/565 and 620 nm (±GR) based on EEM by MM type (a and b), MM pT stage (c and d), MM ulceration (e and f), MM melan A (f and g).

Figure 5 Emission fluorescence at 565 nm divided by MM type and MM pT stage.
Figure 5

Emission fluorescence at 565 nm divided by MM type and MM pT stage.

EEM of urine and healthy controls were characterised by four endogenous fluorescent regions (Figure 6a). Fluorescence maximum marked as A (λEX = 340; λEM = 410 nm) after addition of GR could represent the fluorescence of elastin/collagens and elastin/collagen fragments, fluorescence maximum marked as B (λEX = 340 nm; λEM = 440–460 nm) could represent the endogenous fluorophore-free NADH and maximum marked as C could either represent bilirubin and other lipoid components (found at λEX = 370–380 nm; λEM = 490–510 nm) or it could be lipofuscin and lipofuscin-like lipopigments (ceroids) (found at λEX = 400–500 nm; λEM = 480–700 nm). The isolated maximum marked as D (λEX = 450 nm, λEM = 520 nm) can be attributed to the fluorescence of flavins (FAD) and their metabolites. Some urine specimens of patients also exhibited a further maximum marked as E, consisting of three further peaks (E1–E3) (λEX = 390–400 nm; λEM = 565, 620, 680 nm), which may have resulted from endogenous fluorescence of lipofuscin, and its derivative luminescent ferrous clusters (porphyrins), or bilirubin.

Figure 6 (a) EEM with denoted maxima; (b) EEM MM urine; (c) EEM MM urine + GR; (d) EEM control urine; (e) EEM control urine + GR.
Figure 6

(a) EEM with denoted maxima; (b) EEM MM urine; (c) EEM MM urine + GR; (d) EEM control urine; (e) EEM control urine + GR.

When comparing the EEM of the control group and MM patients, there was specific decrease of fluorescence intensity after the addition of GR to the urine sample prior to the analysis. These changes were mainly present in the excitation region of 350–460 nm and in the emission region of 500–650 nm (Figure 6b and d). In the control group, the decrease of fluorescence after GR addition was not as pronounced in comparison with MM patients at above-mentioned emission wavelengths (Figure 6c and e).

The level of measured endogenous fluorophores in urine reflects not only the metabolic but also the genetic state of MM cells. Genes which are directly or indirectly involved in energetic and pigment metabolism of MM cells were analysed. The gene expression of HIF-1α, MTF-M and IGF-1 (Figure 7a–f) for single pT stages of MM and for ulcerated and non-ulcerated MM corresponds with malignancy of the disease. The average values of relative gene expression and p values of significance of unpaired t-test and F-test of variances are in Table 3. The ordinary one-way ANOVA for HIF-1α expression between groups of pT stages resulted in significance with the p level 0.038, for MITF-M the significance was with p = 0.003 and for IGF1 the significance was with p < 0.0001. The Brown–Forsytlett’s test for HIF-1α was non-significant with the p value 0.4829, for MITF-M the test significance was with the p value 0.0120, and for IGF1 test was non-significant with p = 0.102. The two-way ANOVA Dunnett’s multiple comparisons test showed significance change for comparing MITF-M gene expression in the pT1ab stage with the p value 0.006 and the pT2ab stage with p = 0.002, and if comparing IGF1 for pT1ab with the p = 0.021, pT2ab with p = 0.027, pT3ab with p < 0.0001, pT4ab with p < 0.001 and undefined stage with p = 0.003. The rest of gene expression for HIF-1α and MITF-M was non-significant (Table 3).

Figure 7 Gene expression by MM pT stage and MM ulceration of HIF-1α (a and b), MITF-M (c and d), IGF1 (e and f).
Figure 7

Gene expression by MM pT stage and MM ulceration of HIF-1α (a and b), MITF-M (c and d), IGF1 (e and f).

Table 3

Gene expression and p values of gene expression compared to controls

Relative gene expression
pT1abpT2abpT3abpT4abUndefinedControls
HIF-1α0.430.370.710.780.570.20
MITF-M0.290.210.810.790.601.13
IGF13.103.045.435.073.561.12
Unpaired t-test two-tailed
pT1abpT2abpT3abpT4abUndefined
HIF-1α0.190.200.00030.0040.07
MITF-M0.010.0040.250.400.08
IGF10.00020.003<0.0001<0.00010.0002
F-test of variances
pT1abpT2abpT3abpT4abUndefined
HIF-1α0.050.850.520.770.01
MITF-M0.660.020.080.080.15
IGF1<0.0001<0.00010.00220.001<0.0001
One-way ANOVA Dunnett’s multiple comparisons test
pT1abpT2abpT3abpT4abUndefined
HIF-1α0.640.850.020.080.19
MITF-M0.010.0020.560.770.14
IGF10.020.03<0.00010.00020.003

4 Discussion

MM is one of the most aggressive forms of cancer. Neoplastic changes in skin cells are predominantly due to physical damage, such as an inappropriate exposure to UV radiation (mainly UVc) [44], which damages the genetic information of healthy melanocytes and leads to tumour transformation (UVb radiation is required to activate provitamin D in the dermis). Repeated sunburn caused by inadequate exposure to sunlight, and therefore UV radiation, increases the risk of MM. Other risk factors include skin phototype (mainly I and II) [45], lifestyle (low frequency of exposure to sunlight during the year) or genetic predisposition (familial occurrence and numerous benign skin tumours). The Caucasian race, mainly people living in the countryside, suffers predominantly from hypovitaminosis D and has light skin phototypes, and in developed industrial countries lifestyle (avoidance of direct sunlight) leads to more frequent skin malignancies [46,47,48]. Early diagnosis of the disease would certainly contribute to a reduction in MM mortality. Fluorescence methods utilising the luminescence of endogenous fluorophores characteristic for MM (e.g. lipofuscin) have still unexplored potential in detection of both presence and predisposition of MM.

The absorption spectra were characterised by decrease maxima at 280 nm which represent total value of proteins and amino acids [49]. Next, the spectra showed increase at λAB = 340 nm (could correspond with NADH) [50], λAB = 375 nm and λAB = 450 nm (could present FAD) [51]. This change corresponds with presence of cancer disease [52].

As mentioned above, in the urine samples of MM patients and healthy controls, we detected various biomarkers of cancer diseases by fluorescence spectrophotometry. In excitation spectra, we mainly focused on area between 340 and 450 nm where could be detected, for example, NADH (340 nm) [53], elastin and collagens (340–360 nm) [54] and FAD (450 nm) [55], and their mutual ratio. The REX = 450/365 ratio was 0.8871 for the control group and 0.7422 for patients, thus 1.2-fold higher relative to patients. Change in the intensity at 365 nm in MM samples may reflect a lower relative protein concentration (elastin, collagens) as a result of malignant skin disease [56,57], and increased intensity at 340 nm reflects concentration of free NADH or NADPH, respectively [58]. NADH is used by GR to convert GSSG to reduced GSH [43]. This convert could be used for calculation of the NAD(P)H value in samples. Values of the difference of emitted fluorescence signal at 440 and 460 nm (after excitation by λEX = 340 nm) before and after addition of GR reveal a difference between the patients (96% or 51%, respectively) and controls (0.15%).

Fluorescence spectra after excitation at λEX = 340 nm exhibit one large peak (410–460 nm) [53]. Several emission maxima such as NADH, elastin and collagens may be present in the described region. Moreover, the intensity of one fluorophore can mask other fluorophores in neighbouring region; therefore, a reduction of the NADH fluorescence signal by GR can be used to highlight other endogenous fluorophores emitting in the given region (350–730 nm). In this way, we could describe the presence probably of FAD, lipofuscin and porphyrins, the relative concentration of which was higher in patients than in healthy controls.

The metabolites in urine reflect the metabolic and genetic state of cells. We focused on genes which are involved in mitochondria metabolism. The genes such as HIF-1α, MITF-M and IGF1 are directly involved in energy metabolism of MM. The pathological changes by malignant transformation of melanocytes give specific alternations in trash metabolites excluded by urine. The increased expression of HIF-1α leads to decreased OXPHOS, preferred glycolysis and increased NADH values [59,60]. We determined increasing expression of HIF-1α with increasing malignancy (relative gene expression for pT1ab was 2.18 times higher than for the controls, pT2ab was 1.89 times higher, pT3ab was 3.60 times higher, pT4ab was 3.96 times higher, undefined pT stage was 2.91 times higher). The same tendency was observed by other groups [61,62,63,64].

The MITF-M role is to regulate the cellular response to ROS stress. The low level of MITF-M leads to the worst fight with a damage caused by ROS [65,66]. It is well known that increased ROS production directs cells to oncogenic transformation [67]. In our experiments, we detected decreased level of MITF-M in all pT stages of MM. Downregulation of MITF-M gene tends to accumulation of lipofuscin [68] as the side effect of melanin degradation in the melanocytes caused by low activity of tyrosinase due to high level of ROS and HIF-1α. Taken together, the low MITF-M expression correlates with increased presentation of lipofuscin in body fluids as well as in urine [69,70].

The last of studied genes was IGF1, known as mitochondrial protector, which protects cells from oxidative stress and regulates normal cell growth [71]. Its pathologically increased expression activates anti-apoptic genes which are members of oncogenes (BCL-2, BCL-X(L)) [33]. We determined increased expression of IGF1 gene in all pT stages of MM.

As we mentioned above, the differences in fluorescence intensities at λEM = 440, 460, 525, 565 nm between groups of type of MM, pT stages, melan A presentation or ulceration respectively is possible to use as marker of prediction of MM. This trend of increased NADH level occurs in early pT stages; therefore, the early pT stages still “work” on glucose TCA which is uncoupled and leads to accumulation of NADH. The pT4ab stage was described by lower level of NADH caused by glutamine TCA metabolism which compensates ROS production in mitochondria and regenerates NAD+ [27,28]. “Lipofuscin” emission intensities divided by pT stages showed their increased values with increasing malignancy of MM.

In the end, in routine operation, technicians encounter enormous amounts of the biological material. Therefore, the measurement of concentration matrices is time-consuming. We managed to eliminate the effect of NADH using GR and thus visualise characteristic peaks (mostly suited for lipoid pigments, porphyrins, elastin and others) regardless of their initial concentration in 50–96% of all analysed urine samples of MM patients. This trend was not observed in the urine samples of healthy subjects. All determined parameters based on excitation and emission spectra of urine discussed above, λAB (280, 340, 375, 450 nm), REX (365, 450 nm), λEM (440–460, 565 nm), show a potential in laboratory diagnostics for monitoring of MM progression. Since our findings correlate with gene expression of HIF-1α, MITF-M and IGF1, potential changes in metabolic pathways associated with MM seem to be directly reflected in both excitation and emission spectra of urine samples. Therefore, spectral measurements of urine samples appear to be a rapid, inexpensive and non-invasive method suitable for monitoring of MM progression.

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Acknowledgements

The data presented were generated in one PhD thesis. The work was supported by the VEGA 1/0620/19.

  1. Conflict of interest: Authors declare no conflict of interest.

  2. Ethical approval: We declare that the study was assessed and approved by the institutional ethics committee/institutional review board and that the letter of approval is available for examination. Permission from ethics committee was issued by Pavol Jozef Šafárik University in Košice, Faculty of Medicine (20N/2016).

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Received: 2020-01-27
Revised: 2020-06-02
Accepted: 2020-06-07
Published Online: 2020-08-07

© 2020 Ivana Špaková et al., published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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  79. The combined use of GC, PDSC and FT-IR techniques to characterize fat extracted from commercial complete dry pet food for adult cats
  80. MALDI-TOF MS profiling in the discovery and identification of salivary proteomic patterns of temporomandibular joint disorders
  81. Concentrations of dioxins, furans and dioxin-like PCBs in natural animal feed additives
  82. Structure and some physicochemical and functional properties of water treated under ammonia with low-temperature low-pressure glow plasma of low frequency
  83. Mesoscale nanoparticles encapsulated with emodin for targeting antifibrosis in animal models
  84. Amine-functionalized magnetic activated carbon as an adsorbent for preconcentration and determination of acidic drugs in environmental water samples using HPLC-DAD
  85. Antioxidant activity as a response to cadmium pollution in three durum wheat genotypes differing in salt-tolerance
  86. A promising naphthoquinone [8-hydroxy-2-(2-thienylcarbonyl)naphtho[2,3-b]thiophene-4,9-dione] exerts anti-colorectal cancer activity through ferroptosis and inhibition of MAPK signaling pathway based on RNA sequencing
  87. Synthesis and efficacy of herbicidal ionic liquids with chlorsulfuron as the anion
  88. Effect of isovalent substitution on the crystal structure and properties of two-slab indates BaLa2−xSmxIn2O7
  89. Synthesis, spectral and thermo-kinetics explorations of Schiff-base derived metal complexes
  90. An improved reduction method for phase stability testing in the single-phase region
  91. Comparative analysis of chemical composition of some commercially important fishes with an emphasis on various Malaysian diets
  92. Development of a solventless stir bar sorptive extraction/thermal desorption large volume injection capillary gas chromatographic-mass spectrometric method for ultra-trace determination of pyrethroids pesticides in river and tap water samples
  93. A turbidity sensor development based on NL-PI observers: Experimental application to the control of a Sinaloa’s River Spirulina maxima cultivation
  94. Deep desulfurization of sintering flue gas in iron and steel works based on low-temperature oxidation
  95. Investigations of metallic elements and phenolics in Chinese medicinal plants
  96. Influence of site-classification approach on geochemical background values
  97. Effects of ageing on the surface characteristics and Cu(ii) adsorption behaviour of rice husk biochar in soil
  98. Adsorption and sugarcane-bagasse-derived activated carbon-based mitigation of 1-[2-(2-chloroethoxy)phenyl]sulfonyl-3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl) urea-contaminated soils
  99. Antimicrobial and antifungal activities of bifunctional cooper(ii) complexes with non-steroidal anti-inflammatory drugs, flufenamic, mefenamic and tolfenamic acids and 1,10-phenanthroline
  100. Application of selenium and silicon to alleviate short-term drought stress in French marigold (Tagetes patula L.) as a model plant species
  101. Screening and analysis of xanthine oxidase inhibitors in jute leaves and their protective effects against hydrogen peroxide-induced oxidative stress in cells
  102. Synthesis and physicochemical studies of a series of mixed-ligand transition metal complexes and their molecular docking investigations against Coronavirus main protease
  103. A study of in vitro metabolism and cytotoxicity of mephedrone and methoxetamine in human and pig liver models using GC/MS and LC/MS analyses
  104. A new phenyl alkyl ester and a new combretin triterpene derivative from Combretum fragrans F. Hoffm (Combretaceae) and antiproliferative activity
  105. Erratum
  106. Erratum to: A one-step incubation ELISA kit for rapid determination of dibutyl phthalate in water, beverage and liquor
  107. Review Articles
  108. Sinoporphyrin sodium, a novel sensitizer for photodynamic and sonodynamic therapy
  109. Natural products isolated from Casimiroa
  110. Plant description, phytochemical constituents and bioactivities of Syzygium genus: A review
  111. Evaluation of elastomeric heat shielding materials as insulators for solid propellant rocket motors: A short review
  112. Special Issue on Applied Biochemistry and Biotechnology 2019
  113. An overview of Monascus fermentation processes for monacolin K production
  114. Study on online soft sensor method of total sugar content in chlorotetracycline fermentation tank
  115. Studies on the Anti-Gouty Arthritis and Anti-hyperuricemia Properties of Astilbin in Animal Models
  116. Effects of organic fertilizer on water use, photosynthetic characteristics, and fruit quality of pear jujube in northern Shaanxi
  117. Characteristics of the root exudate release system of typical plants in plateau lakeside wetland under phosphorus stress conditions
  118. Characterization of soil water by the means of hydrogen and oxygen isotope ratio at dry-wet season under different soil layers in the dry-hot valley of Jinsha River
  119. Composition and diurnal variation of floral scent emission in Rosa rugosa Thunb. and Tulipa gesneriana L.
  120. Preparation of a novel ginkgolide B niosomal composite drug
  121. The degradation, biodegradability and toxicity evaluation of sulfamethazine antibiotics by gamma radiation
  122. Special issue on Monitoring, Risk Assessment and Sustainable Management for the Exposure to Environmental Toxins
  123. Insight into the cadmium and zinc binding potential of humic acids derived from composts by EEM spectra combined with PARAFAC analysis
  124. Source apportionment of soil contamination based on multivariate receptor and robust geostatistics in a typical rural–urban area, Wuhan city, middle China
  125. Special Issue on 13th JCC 2018
  126. The Role of H2C2O4 and Na2CO3 as Precipitating Agents on The Physichochemical Properties and Photocatalytic Activity of Bismuth Oxide
  127. Preparation of magnetite-silica–cetyltrimethylammonium for phenol removal based on adsolubilization
  128. Topical Issue on Agriculture
  129. Size-dependent growth kinetics of struvite crystals in wastewater with calcium ions
  130. The effect of silica-calcite sedimentary rock contained in the chicken broiler diet on the overall quality of chicken muscles
  131. Physicochemical properties of selected herbicidal products containing nicosulfuron as an active ingredient
  132. Lycopene in tomatoes and tomato products
  133. Fluorescence in the assessment of the share of a key component in the mixing of feed
  134. Sulfur application alleviates chromium stress in maize and wheat
  135. Effectiveness of removal of sulphur compounds from the air after 3 years of biofiltration with a mixture of compost soil, peat, coconut fibre and oak bark
  136. Special Issue on the 4th Green Chemistry 2018
  137. Study and fire test of banana fibre reinforced composites with flame retardance properties
  138. Special Issue on the International conference CosCI 2018
  139. Disintegration, In vitro Dissolution, and Drug Release Kinetics Profiles of k-Carrageenan-based Nutraceutical Hard-shell Capsules Containing Salicylamide
  140. Synthesis of amorphous aluminosilicate from impure Indonesian kaolin
  141. Special Issue on the International Conf on Science, Applied Science, Teaching and Education 2019
  142. Functionalization of Congo red dye as a light harvester on solar cell
  143. The effect of nitrite food preservatives added to se’i meat on the expression of wild-type p53 protein
  144. Biocompatibility and osteoconductivity of scaffold porous composite collagen–hydroxyapatite based coral for bone regeneration
  145. Special Issue on the Joint Science Congress of Materials and Polymers (ISCMP 2019)
  146. Effect of natural boron mineral use on the essential oil ratio and components of Musk Sage (Salvia sclarea L.)
  147. A theoretical and experimental study of the adsorptive removal of hexavalent chromium ions using graphene oxide as an adsorbent
  148. A study on the bacterial adhesion of Streptococcus mutans in various dental ceramics: In vitro study
  149. Corrosion study of copper in aqueous sulfuric acid solution in the presence of (2E,5E)-2,5-dibenzylidenecyclopentanone and (2E,5E)-bis[(4-dimethylamino)benzylidene]cyclopentanone: Experimental and theoretical study
  150. Special Issue on Chemistry Today for Tomorrow 2019
  151. Diabetes mellitus type 2: Exploratory data analysis based on clinical reading
  152. Multivariate analysis for the classification of copper–lead and copper–zinc glasses
  153. Special Issue on Advances in Chemistry and Polymers
  154. The spatial and temporal distribution of cationic and anionic radicals in early embryo implantation
  155. Special Issue on 3rd IC3PE 2020
  156. Magnetic iron oxide/clay nanocomposites for adsorption and catalytic oxidation in water treatment applications
  157. Special Issue on IC3PE 2018/2019 Conference
  158. Exergy analysis of conventional and hydrothermal liquefaction–esterification processes of microalgae for biodiesel production
  159. Advancing biodiesel production from microalgae Spirulina sp. by a simultaneous extraction–transesterification process using palm oil as a co-solvent of methanol
  160. Topical Issue on Applications of Mathematics in Chemistry
  161. Omega and the related counting polynomials of some chemical structures
  162. M-polynomial and topological indices of zigzag edge coronoid fused by starphene
https://doi.org/10.1515/chem-2020-0143
Schlagwörter für diesen Artikel
NAD(P)H; lipofuscin; HIF-1α; MITF-M; IGF1
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Artikel in diesem Heft

  1. Regular Articles
  2. Electrochemical antioxidant screening and evaluation based on guanine and chitosan immobilized MoS2 nanosheet modified glassy carbon electrode (guanine/CS/MoS2/GCE)
  3. Kinetic models of the extraction of vanillic acid from pumpkin seeds
  4. On the maximum ABC index of bipartite graphs without pendent vertices
  5. Estimation of the total antioxidant potential in the meat samples using thin-layer chromatography
  6. Molecular dynamics simulation of sI methane hydrate under compression and tension
  7. Spatial distribution and potential ecological risk assessment of some trace elements in sediments and grey mangrove (Avicennia marina) along the Arabian Gulf coast, Saudi Arabia
  8. Amino-functionalized graphene oxide for Cr(VI), Cu(II), Pb(II) and Cd(II) removal from industrial wastewater
  9. Chemical composition and in vitro activity of Origanum vulgare L., Satureja hortensis L., Thymus serpyllum L. and Thymus vulgaris L. essential oils towards oral isolates of Candida albicans and Candida glabrata
  10. Effect of excess Fluoride consumption on Urine-Serum Fluorides, Dental state and Thyroid Hormones among children in “Talab Sarai” Punjab Pakistan
  11. Design, Synthesis and Characterization of Novel Isoxazole Tagged Indole Hybrid Compounds
  12. Comparison of kinetic and enzymatic properties of intracellular phosphoserine aminotransferases from alkaliphilic and neutralophilic bacteria
  13. Green Organic Solvent-Free Oxidation of Alkylarenes with tert-Butyl Hydroperoxide Catalyzed by Water-Soluble Copper Complex
  14. Ducrosia ismaelis Asch. essential oil: chemical composition profile and anticancer, antimicrobial and antioxidant potential assessment
  15. DFT calculations as an efficient tool for prediction of Raman and infra-red spectra and activities of newly synthesized cathinones
  16. Influence of Chemical Osmosis on Solute Transport and Fluid Velocity in Clay Soils
  17. A New fatty acid and some triterpenoids from propolis of Nkambe (North-West Region, Cameroon) and evaluation of the antiradical scavenging activity of their extracts
  18. Antiplasmodial Activity of Stigmastane Steroids from Dryobalanops oblongifolia Stem Bark
  19. Rapid identification of direct-acting pancreatic protectants from Cyclocarya paliurus leaves tea by the method of serum pharmacochemistry combined with target cell extraction
  20. Immobilization of Pseudomonas aeruginosa static biomass on eggshell powder for on-line preconcentration and determination of Cr (VI)
  21. Assessment of methyl 2-({[(4,6-dimethoxypyrimidin-2-yl)carbamoyl] sulfamoyl}methyl)benzoate through biotic and abiotic degradation modes
  22. Stability of natural polyphenol fisetin in eye drops Stability of fisetin in eye drops
  23. Production of a bioflocculant by using activated sludge and its application in Pb(II) removal from aqueous solution
  24. Molecular Properties of Carbon Crystal Cubic Structures
  25. Synthesis and characterization of calcium carbonate whisker from yellow phosphorus slag
  26. Study on the interaction between catechin and cholesterol by the density functional theory
  27. Analysis of some pharmaceuticals in the presence of their synthetic impurities by applying hybrid micelle liquid chromatography
  28. Two mixed-ligand coordination polymers based on 2,5-thiophenedicarboxylic acid and flexible N-donor ligands: the protective effect on periodontitis via reducing the release of IL-1β and TNF-α
  29. Incorporation of silver stearate nanoparticles in methacrylate polymeric monoliths for hemeprotein isolation
  30. Development of ultrasound-assisted dispersive solid-phase microextraction based on mesoporous carbon coated with silica@iron oxide nanocomposite for preconcentration of Te and Tl in natural water systems
  31. N,N′-Bis[2-hydroxynaphthylidene]/[2-methoxybenzylidene]amino]oxamides and their divalent manganese complexes: Isolation, spectral characterization, morphology, antibacterial and cytotoxicity against leukemia cells
  32. Determination of the content of selected trace elements in Polish commercial fruit juices and health risk assessment
  33. Diorganotin(iv) benzyldithiocarbamate complexes: synthesis, characterization, and thermal and cytotoxicity study
  34. Keratin 17 is induced in prurigo nodularis lesions
  35. Anticancer, antioxidant, and acute toxicity studies of a Saudi polyherbal formulation, PHF5
  36. LaCoO3 perovskite-type catalysts in syngas conversion
  37. Comparative studies of two vegetal extracts from Stokesia laevis and Geranium pratense: polyphenol profile, cytotoxic effect and antiproliferative activity
  38. Fragmentation pattern of certain isatin–indole antiproliferative conjugates with application to identify their in vitro metabolic profiles in rat liver microsomes by liquid chromatography tandem mass spectrometry
  39. Investigation of polyphenol profile, antioxidant activity and hepatoprotective potential of Aconogonon alpinum (All.) Schur roots
  40. Lead discovery of a guanidinyl tryptophan derivative on amyloid cascade inhibition
  41. Physicochemical evaluation of the fruit pulp of Opuntia spp growing in the Mediterranean area under hard climate conditions
  42. Electronic structural properties of amino/hydroxyl functionalized imidazolium-based bromide ionic liquids
  43. New Schiff bases of 2-(quinolin-8-yloxy)acetohydrazide and their Cu(ii), and Zn(ii) metal complexes: their in vitro antimicrobial potentials and in silico physicochemical and pharmacokinetics properties
  44. Treatment of adhesions after Achilles tendon injury using focused ultrasound with targeted bFGF plasmid-loaded cationic microbubbles
  45. Synthesis of orotic acid derivatives and their effects on stem cell proliferation
  46. Chirality of β2-agonists. An overview of pharmacological activity, stereoselective analysis, and synthesis
  47. Fe3O4@urea/HITh-SO3H as an efficient and reusable catalyst for the solvent-free synthesis of 7-aryl-8H-benzo[h]indeno[1,2-b]quinoline-8-one and indeno[2′,1′:5,6]pyrido[2,3-d]pyrimidine derivatives
  48. Adsorption kinetic characteristics of molybdenum in yellow-brown soil in response to pH and phosphate
  49. Enhancement of thermal properties of bio-based microcapsules intended for textile applications
  50. Exploring the effect of khat (Catha edulis) chewing on the pharmacokinetics of the antiplatelet drug clopidogrel in rats using the newly developed LC-MS/MS technique
  51. A green strategy for obtaining anthraquinones from Rheum tanguticum by subcritical water
  52. Cadmium (Cd) chloride affects the nutrient uptake and Cd-resistant bacterium reduces the adsorption of Cd in muskmelon plants
  53. Removal of H2S by vermicompost biofilter and analysis on bacterial community
  54. Structural cytotoxicity relationship of 2-phenoxy(thiomethyl)pyridotriazolopyrimidines: Quantum chemical calculations and statistical analysis
  55. A self-breaking supramolecular plugging system as lost circulation material in oilfield
  56. Synthesis, characterization, and pharmacological evaluation of thiourea derivatives
  57. Application of drug–metal ion interaction principle in conductometric determination of imatinib, sorafenib, gefitinib and bosutinib
  58. Synthesis and characterization of a novel chitosan-grafted-polyorthoethylaniline biocomposite and utilization for dye removal from water
  59. Optimisation of urine sample preparation for shotgun proteomics
  60. DFT investigations on arylsulphonyl pyrazole derivatives as potential ligands of selected kinases
  61. Treatment of Parkinson’s disease using focused ultrasound with GDNF retrovirus-loaded microbubbles to open the blood–brain barrier
  62. New derivatives of a natural nordentatin
  63. Fluorescence biomarkers of malignant melanoma detectable in urine
  64. Study of the remediation effects of passivation materials on Pb-contaminated soil
  65. Saliva proteomic analysis reveals possible biomarkers of renal cell carcinoma
  66. Withania frutescens: Chemical characterization, analgesic, anti-inflammatory, and healing activities
  67. Design, synthesis and pharmacological profile of (−)-verbenone hydrazones
  68. Synthesis of magnesium carbonate hydrate from natural talc
  69. Stability-indicating HPLC-DAD assay for simultaneous quantification of hydrocortisone 21 acetate, dexamethasone, and fluocinolone acetonide in cosmetics
  70. A novel lactose biosensor based on electrochemically synthesized 3,4-ethylenedioxythiophene/thiophene (EDOT/Th) copolymer
  71. Citrullus colocynthis (L.) Schrad: Chemical characterization, scavenging and cytotoxic activities
  72. Development and validation of a high performance liquid chromatography/diode array detection method for estrogen determination: Application to residual analysis in meat products
  73. PCSK9 concentrations in different stages of subclinical atherosclerosis and their relationship with inflammation
  74. Development of trace analysis for alkyl methanesulfonates in the delgocitinib drug substance using GC-FID and liquid–liquid extraction with ionic liquid
  75. Electrochemical evaluation of the antioxidant capacity of natural compounds on glassy carbon electrode modified with guanine-, polythionine-, and nitrogen-doped graphene
  76. A Dy(iii)–organic framework as a fluorescent probe for highly selective detection of picric acid and treatment activity on human lung cancer cells
  77. A Zn(ii)–organic cage with semirigid ligand for solvent-free cyanosilylation and inhibitory effect on ovarian cancer cell migration and invasion ability via regulating mi-RNA16 expression
  78. Polyphenol content and antioxidant activities of Prunus padus L. and Prunus serotina L. leaves: Electrochemical and spectrophotometric approach and their antimicrobial properties
  79. The combined use of GC, PDSC and FT-IR techniques to characterize fat extracted from commercial complete dry pet food for adult cats
  80. MALDI-TOF MS profiling in the discovery and identification of salivary proteomic patterns of temporomandibular joint disorders
  81. Concentrations of dioxins, furans and dioxin-like PCBs in natural animal feed additives
  82. Structure and some physicochemical and functional properties of water treated under ammonia with low-temperature low-pressure glow plasma of low frequency
  83. Mesoscale nanoparticles encapsulated with emodin for targeting antifibrosis in animal models
  84. Amine-functionalized magnetic activated carbon as an adsorbent for preconcentration and determination of acidic drugs in environmental water samples using HPLC-DAD
  85. Antioxidant activity as a response to cadmium pollution in three durum wheat genotypes differing in salt-tolerance
  86. A promising naphthoquinone [8-hydroxy-2-(2-thienylcarbonyl)naphtho[2,3-b]thiophene-4,9-dione] exerts anti-colorectal cancer activity through ferroptosis and inhibition of MAPK signaling pathway based on RNA sequencing
  87. Synthesis and efficacy of herbicidal ionic liquids with chlorsulfuron as the anion
  88. Effect of isovalent substitution on the crystal structure and properties of two-slab indates BaLa2−xSmxIn2O7
  89. Synthesis, spectral and thermo-kinetics explorations of Schiff-base derived metal complexes
  90. An improved reduction method for phase stability testing in the single-phase region
  91. Comparative analysis of chemical composition of some commercially important fishes with an emphasis on various Malaysian diets
  92. Development of a solventless stir bar sorptive extraction/thermal desorption large volume injection capillary gas chromatographic-mass spectrometric method for ultra-trace determination of pyrethroids pesticides in river and tap water samples
  93. A turbidity sensor development based on NL-PI observers: Experimental application to the control of a Sinaloa’s River Spirulina maxima cultivation
  94. Deep desulfurization of sintering flue gas in iron and steel works based on low-temperature oxidation
  95. Investigations of metallic elements and phenolics in Chinese medicinal plants
  96. Influence of site-classification approach on geochemical background values
  97. Effects of ageing on the surface characteristics and Cu(ii) adsorption behaviour of rice husk biochar in soil
  98. Adsorption and sugarcane-bagasse-derived activated carbon-based mitigation of 1-[2-(2-chloroethoxy)phenyl]sulfonyl-3-(4-methoxy-6-methyl-1,3,5-triazin-2-yl) urea-contaminated soils
  99. Antimicrobial and antifungal activities of bifunctional cooper(ii) complexes with non-steroidal anti-inflammatory drugs, flufenamic, mefenamic and tolfenamic acids and 1,10-phenanthroline
  100. Application of selenium and silicon to alleviate short-term drought stress in French marigold (Tagetes patula L.) as a model plant species
  101. Screening and analysis of xanthine oxidase inhibitors in jute leaves and their protective effects against hydrogen peroxide-induced oxidative stress in cells
  102. Synthesis and physicochemical studies of a series of mixed-ligand transition metal complexes and their molecular docking investigations against Coronavirus main protease
  103. A study of in vitro metabolism and cytotoxicity of mephedrone and methoxetamine in human and pig liver models using GC/MS and LC/MS analyses
  104. A new phenyl alkyl ester and a new combretin triterpene derivative from Combretum fragrans F. Hoffm (Combretaceae) and antiproliferative activity
  105. Erratum
  106. Erratum to: A one-step incubation ELISA kit for rapid determination of dibutyl phthalate in water, beverage and liquor
  107. Review Articles
  108. Sinoporphyrin sodium, a novel sensitizer for photodynamic and sonodynamic therapy
  109. Natural products isolated from Casimiroa
  110. Plant description, phytochemical constituents and bioactivities of Syzygium genus: A review
  111. Evaluation of elastomeric heat shielding materials as insulators for solid propellant rocket motors: A short review
  112. Special Issue on Applied Biochemistry and Biotechnology 2019
  113. An overview of Monascus fermentation processes for monacolin K production
  114. Study on online soft sensor method of total sugar content in chlorotetracycline fermentation tank
  115. Studies on the Anti-Gouty Arthritis and Anti-hyperuricemia Properties of Astilbin in Animal Models
  116. Effects of organic fertilizer on water use, photosynthetic characteristics, and fruit quality of pear jujube in northern Shaanxi
  117. Characteristics of the root exudate release system of typical plants in plateau lakeside wetland under phosphorus stress conditions
  118. Characterization of soil water by the means of hydrogen and oxygen isotope ratio at dry-wet season under different soil layers in the dry-hot valley of Jinsha River
  119. Composition and diurnal variation of floral scent emission in Rosa rugosa Thunb. and Tulipa gesneriana L.
  120. Preparation of a novel ginkgolide B niosomal composite drug
  121. The degradation, biodegradability and toxicity evaluation of sulfamethazine antibiotics by gamma radiation
  122. Special issue on Monitoring, Risk Assessment and Sustainable Management for the Exposure to Environmental Toxins
  123. Insight into the cadmium and zinc binding potential of humic acids derived from composts by EEM spectra combined with PARAFAC analysis
  124. Source apportionment of soil contamination based on multivariate receptor and robust geostatistics in a typical rural–urban area, Wuhan city, middle China
  125. Special Issue on 13th JCC 2018
  126. The Role of H2C2O4 and Na2CO3 as Precipitating Agents on The Physichochemical Properties and Photocatalytic Activity of Bismuth Oxide
  127. Preparation of magnetite-silica–cetyltrimethylammonium for phenol removal based on adsolubilization
  128. Topical Issue on Agriculture
  129. Size-dependent growth kinetics of struvite crystals in wastewater with calcium ions
  130. The effect of silica-calcite sedimentary rock contained in the chicken broiler diet on the overall quality of chicken muscles
  131. Physicochemical properties of selected herbicidal products containing nicosulfuron as an active ingredient
  132. Lycopene in tomatoes and tomato products
  133. Fluorescence in the assessment of the share of a key component in the mixing of feed
  134. Sulfur application alleviates chromium stress in maize and wheat
  135. Effectiveness of removal of sulphur compounds from the air after 3 years of biofiltration with a mixture of compost soil, peat, coconut fibre and oak bark
  136. Special Issue on the 4th Green Chemistry 2018
  137. Study and fire test of banana fibre reinforced composites with flame retardance properties
  138. Special Issue on the International conference CosCI 2018
  139. Disintegration, In vitro Dissolution, and Drug Release Kinetics Profiles of k-Carrageenan-based Nutraceutical Hard-shell Capsules Containing Salicylamide
  140. Synthesis of amorphous aluminosilicate from impure Indonesian kaolin
  141. Special Issue on the International Conf on Science, Applied Science, Teaching and Education 2019
  142. Functionalization of Congo red dye as a light harvester on solar cell
  143. The effect of nitrite food preservatives added to se’i meat on the expression of wild-type p53 protein
  144. Biocompatibility and osteoconductivity of scaffold porous composite collagen–hydroxyapatite based coral for bone regeneration
  145. Special Issue on the Joint Science Congress of Materials and Polymers (ISCMP 2019)
  146. Effect of natural boron mineral use on the essential oil ratio and components of Musk Sage (Salvia sclarea L.)
  147. A theoretical and experimental study of the adsorptive removal of hexavalent chromium ions using graphene oxide as an adsorbent
  148. A study on the bacterial adhesion of Streptococcus mutans in various dental ceramics: In vitro study
  149. Corrosion study of copper in aqueous sulfuric acid solution in the presence of (2E,5E)-2,5-dibenzylidenecyclopentanone and (2E,5E)-bis[(4-dimethylamino)benzylidene]cyclopentanone: Experimental and theoretical study
  150. Special Issue on Chemistry Today for Tomorrow 2019
  151. Diabetes mellitus type 2: Exploratory data analysis based on clinical reading
  152. Multivariate analysis for the classification of copper–lead and copper–zinc glasses
  153. Special Issue on Advances in Chemistry and Polymers
  154. The spatial and temporal distribution of cationic and anionic radicals in early embryo implantation
  155. Special Issue on 3rd IC3PE 2020
  156. Magnetic iron oxide/clay nanocomposites for adsorption and catalytic oxidation in water treatment applications
  157. Special Issue on IC3PE 2018/2019 Conference
  158. Exergy analysis of conventional and hydrothermal liquefaction–esterification processes of microalgae for biodiesel production
  159. Advancing biodiesel production from microalgae Spirulina sp. by a simultaneous extraction–transesterification process using palm oil as a co-solvent of methanol
  160. Topical Issue on Applications of Mathematics in Chemistry
  161. Omega and the related counting polynomials of some chemical structures
  162. M-polynomial and topological indices of zigzag edge coronoid fused by starphene
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