Gender differences in the fluorescence of human skin in young healthy adults.

BACKGROUND: Human skin naturally contains many endogenous fluorophores; therefore, fluorescence techniques can be used for monitoring of the human skin even in in vivo mode. The aim of this work was to study skin autofluorescence in vivo regarding the possible effect of gender. MATERIALS AND METHODS: Fluorescence emission spectra of young healthy Caucasian adults in 3 anatomical regions (forehead, hand, and inner upper arm) were taken with excitation at 280, 325, or 400 nm. RESULTS: Three emission bands were found in the spectra for both men and women: (1) an intensive band peaked at 340/280 nm (peak emission/excitation wavelength), corresponding to aromatic amino acids of proteins in epidermis; (2) a broad band with emission between 360 nm and 480 nm (excitation 325 nm) with a base peak around 390 nm and 2 side peaks at 420 and 450 nm, mainly due to collagen cross-links in dermis with a possible weak contribution of elastin and mitochondrial NADPH; (3) a weak but distinct peak at 600/400 nm corresponding presumably to skin unmetalled porphyrins. CONCLUSION: The intensity of skin autofluorescence showed differences between genders and among anatomical regions. The 340 nm intensity was 1.4 times higher in the male group in all 3 anatomical regions studied. The highest intensity of skin autofluorescence for the peaks at 340/280 nm and 600/400 nm was found on the forehead, whereas the 390/325 nm band was most intensive on the inner upper arm in both genders.

Urinary fluorescence analysis in diagnosis of bladder cancer.

Early diagnosis of bladder cancer is crucial for improvement of cancer specific survival and recurrence rate. We analyzed the possible role of fluorescence urine analysis in bladder cancer diagnosis. The cohort consisted of 20 healthy controls, 40 patients with hematuria and 75 patients with hematuria and histologically proven bladder tumor. Synchronous fluores- cence spectra with a 70 nm wavelength difference were recorded for (1:1-1:128) urine dilutions. Concentration matrices of synchronous spectra (CMSS) were used to classify samples into tested groups. CMSS analysis allowed us to distinguish patients with tumor from patients with hematuria with a sensitivity 55% and specificity 74.7%. This is comparable to the sensitivity and specificity of other non-invasive tests like BTA stat and nmP-22 (Bladder check®). Lower fluorescence inten- sity of Imax 280 nm and ratio of 280 nm to 450 nm was found to be associated with the presence of tumor. We have found an association of decreased fluorescence with the stage of the disease. Our data suggest that CMSS urine analysis has a potential role in the non-invasive diagnostic tests for bladder cancer, but it cannot replace the current diagnostic algorithm yet.

Differences in pteridine urinary levels in patients with malignant and benign ovarian tumors in comparison with healthy individuals.

Pteridines belong to a class of fluorescent metabolites that are excreted by humans in urine and their concentrations can reflect various pathophysiological states. We quantified the differences in urinary pteridine levels in patients with malignant and benign ovarian tumors and in healthy individuals. Urine samples were centrifuged and supernatants were oxidized by MnO2 before analysis. Levels of neopterin, biopterin, and pterin were assessed by fluorescence analysis of human urine after HPLC separation. We have revealed that the median neopterin levels were higher in urine samples from patients with malignant (0.226 µmol/mmol creatinine) and benign ovarian tumors (0.150 µmol/mmol creatinine) than in healthy subjects (0.056 µmol/mmol creatinine). The median neopterin levels of patients with malignant tumors were higher (1.5-times) than in patients with benign tumors. The median biopterin level in urine of patients with benign ovarian tumors (0.268 µmol/mmol creatinine) was found to be very close to the level in patients with malignant ovarian tumors (0.239 µmol/mmol creatinine), and both were higher than in healthy samples (0.096 µmol/mmol creatinine). The levels of urine pterin followed a pattern similar to neopterin levels for both ovarian tumors, but their concentrations were about three times lower than neopterin levels.

Fluorescence analysis of urine and its potential for ovarian cancer screening.

Early diagnosis of ovarian cancer could lead to decreased mortality. We assessed the possible use of urine autofluorescence analysis in its diagnostics and screening.We analysed urine from 42 healthy volunteers, 35 patients with benign, and 36 patients with malignant ovarian tumors. Synchronous fluorescence spectra with a 70 nm wavelength difference were recorded for (1:1 - 1:1024) urine dilutions. Concentration matrices of synchronous spectra (CMSS) were used to classify samples into tested groups.CMSS analysis allowed us to distinguish patients with malignant tumors from healthy ones with a high sensitivity (91.67 %) and specificity (100 %), a positive predictive value (PPV) 100 % and a negative predictive value (NPV) 93.33 %. However, discrimination between benign and malignant ovarian tumors was weaker, with sensitivity 86.11 %, specificity 77.14 %, PPV 79.49 % and NPV 84.38 %. Fluorescence intensity and the position of peaks at 330 and 360 nm were found to be associated with the grade and stage, suggesting that different fluorescent metabolites may prevail at different stages of the disease.CMSS analysis of urine provides an alternative for ovarian cancer screening method development and could be used as a diagnostic test to detect the recurrence of the disease after therapy.

The effect of vitamin D3 supplementation on intracellular calcium and plasma membrane calcium ATPase activity in early stages of chronic kidney disease.

Chronic kidney disease (CKD) is associated with increased concentration of intracellular calcium, which is pathological and may lead to irreversible damage of cell functions and structures. The aim of our study was to investigate the impact of 6 months vitamin D(3) supplementation (14 000 IU/week) on free cytosolic calcium concentration ([Ca(2+)](i)) and on the plasma membrane calcium ATPase (PMCA) activity of patients with CKD stage 2-3. PMCA activity of patients was also compared to that of healthy volunteers. Vitamin D(3) supplementation of CKD patients resulted in the decrease of [Ca(2+)](i) (119.79+/-5.87 nmol/l vs. 105.36+/-3.59 nmol/l, n=14, P<0.001), whereas PMCA activity of CKD patients (38.75+/-22.89 nmol P(i)/mg/h) remained unchanged after vitamin D(3) supplementation (40.96+/-17.74 nmol P(i)/mg/h, n=14). PMCA activity of early stage CKD patients before supplementation of vitamin D(3), was reduced by 34 % (42.01+/-20.64 nmol P(i)/mg/h) in comparison to healthy volunteers (63.68+/-20.32 nmol P(i)/mg/h, n=28, P<0.001). These results indicate that vitamin D(3) supplementation had a lowering effect on [Ca(2+)](i) and negligible effect on PMCA activity in CKD patients.

Fluorescence characteristics of human urine from normal individuals and ovarian cancer patients.

Early diagnostics of ovarian cancer is difficult, because there are no symptoms until the disease has progressed to an advanced stage. As urine contains many intrinsic fluorophores, modern fluorescence techniques are perspective candidates for new routine urine tests. The presented work deals with differences in the fluorescence of metabolites in urine of ovarian cancer patients comparing to healthy volunteers using the fluorescence excitation-emission matrices. The most serious differences were found in undiluted urine at the fluorescence emission wavelengths from 400 nm to 460 nm when excited at 310 - 390 nm. Statistical analyses of our data have shown a 5-fold reduction in the intensity of the peak at 330/420 nm (excitation/emission wavelength) for undiluted urine samples excreted by cancer patients as compared to those of normal donors. Moreover, the ratio of intensities of the peaks at 370/440 nm and at 330/420 nm is 18-times elevated in urine excreted by patients with ovarian cancer as compared to healthy urine samples. The observed changes could be interpreted as reduction of the presence of pyridoxic acid, whereas blue-fluorescing pteridines becomes dominant in excitation-emission matrices of cancer urine samples in comparison to healthy donors. We suggest pteridines, which are related to cellular metabolism, as suitable candidates for neoplasia-associated fluorescent markers in human urine. Our work showed that monitoring of human urine fluorescent metabolites offers an alternative for ovarian cancer screening.

Involvement of membrane fluidity in endogenous protective processes running on subcellular membrane systems of the rat heart.

Membrane fluidity is a widely recognized biophysical variable that provides information about structural organization of the subcellular membranes exhibiting physical characteristics of liquid crystals. The term "fluidity" reflects in this case the tightness in packing of acyl parts of the membrane phospholipid molecules, a feature that may influence considerably the molecular mobility and via that also the sensitivity and reactivity of membrane-bound transporters, receptors and enzyme systems. Data presented in this review are aimed to demonstrate the substantial role of changes in membrane fluidity occurring in the processes associated with endogenous protection observed in cardiac sarcolemma and mitochondria in diverse pathologies, particularly in diabetes and hypertension.

Effects of chronic kidney disease on blood cells membrane properties.

Chronic kidney disease (CKD) is progressive loss of renal function associated among others with increased intracellular calcium concentration. The purpose of this study was to identify the effects of CKD on cell membrane properties such as human red blood cell Ca(2+) ATPase activity, lymphocyte plasma membrane P2X(7) receptor expression and function. This could help us in elucidating the origin of increased calcium concentration in blood cells. We found out Ca(2+) ATPase activity is decreased in early stage CKD patients resulting in altered calcium removal from cytoplasm. By means of flow cytometry we assessed that P2X(7) receptor expression on lymphocyte membrane is 1.5 fold increased for CKD patients. Moreover, we detected an increased uptake of ethidium bromide through this receptor in CKD at basal conditions. It means CKD lymphocyte membranes contain more receptors which are more permeable thus allowing increased calcium influx from extracellular milieu. Finally, we can state alterations in blood cell membranes are closely linked to CKD and may be responsible for intracellular calcium accumulation.

Liver mitochondrial respiratory function and coenzyme Q content in rats on a hypercholesterolemic diet treated with atorvastatin.

Statins, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, are effective drugs in the treatment of hypercholesterolemia, however, their undesirable actions are not fully known. We investigated the effects of atorvastatin on the oxidative phosphorylation and membrane fluidity in liver mitochondria, and also on the coenzyme Q (CoQ) content in the mitochondria, liver tissue, and plasma of rats on a standard (C) and hypercholesterolemic (HCh) diet. Atorvastatin was administered at either low (10 mg kg(-1)) or high dose (80 mg kg(-1)) for four weeks. The high dose of the drug decreased the concentrations of total cholesterol and triacylglycerols in the plasma and liver of rats on a HCh diet. Administration of atorvastatin was associated with decreased oxygen uptake (state 3), and oxidative phosphorylation rate in the mitochondria of both C and HCh rats. Further, the drug influenced mitochondrial membrane fluidity and dose-dependently reduced concentrations of oxidized and reduced forms of CoQ in the mitochondria. Our findings point to an association between in vivo administration of atorvastatin and impaired bioenergetics in the liver mitochondria of rats, regardless of diet, in conjunction with simultaneous depletion of oxidized and reduced CoQ forms from the mitochondria. This fact may play a significant role in the development of statin-induced hepatopathy.

Penetration of laser light through red blood cell ghosts.

Hemoglobin is the main absorber of visible light in blood and blood-perfused tissues. However, hemoglobin is released from a red blood cell (RBC) during hemolysis. Hemolysis may be caused by a large number of medical conditions, including photodynamic therapy (PDT) and this subsequently can affect passage of light through the treated biological structures. The purpose of the present study was to determine the penetration of a laser beam through a suspension of hemoglobin-free human red blood cells (RBCs) - ghosts. Although hemoglobin has been efficiently removed from the samples used in our experiments, our measurements show that the samples still effectively attenuate the radiant power of penetrating laser light. We established penetration depths of 12.6mm and 15.4mm for two different laser light wavelengths, 532nm and 630nm, respectively. The penetration depth of laser light was about one order of magnitude higher for hemoglobin-free RBC ghosts as compared to intact RBCs [8,10,12]. These results can be important in case of phototherapy or biostimulation, since all photons that penetrate in a biological object may interact with it and evoke biological response.

Endogenous protective mechanisms in remodeling of rat heart mitochondrial membranes in the acute phase of streptozotocin-induced diabetes.

The aim of present study was to investigate functional and physical alterations in membranes of heart mitochondria that are associated with remodeling of these organelles in acute phase of streptozotocin-induced diabetes and to elucidate the role of these changes in adaptation of the heart to acute streptozotocin-induced diabetes (evaluated 8 days after single dose streptozotocin application to male Wistar rats). Action of free radicals on the respiratory chain of diabetic-heart mitochondria was manifested by 17 % increase (p<0.05) in oxidized form of the coenzyme Q(10) and resulted in a decrease of states S3 and S4 respiration, the respiratory control index, rate of phosphorylation (all p<0.01) and the mitochondrial transmembrane potential (p<0.05), but the ADP/O ratio decreased only moderately (p>0.05). On the contrary, membrane fluidity and the total mitochondrial Mg2+-ATPase activity increased (both p<0.05). In diabetic heart mitochondria, linear regression analysis revealed a reciprocal relationship between the increase in membrane fluidity and decrease in trans-membrane potential (p<0.05, r = 0.67). Changes in membrane fluidity, transmembrane potential, Mg2+-ATPase activity and the almost preserved ADP/O ratio appear as the manifestation of endogenous protective mechanisms participating in the functional remodeling of mitochondria which contributes to adaptation of the heart to diabetes.

The response of Na+/K+ -ATPase of human erythrocytes to green laser light treatment.

The objective of this study was to investigate the response of Na(+)/K(+)-ATPase of human erythrocytes to green laser irradiation. Effects of green laser light of fluences 9.5-63.3 J.cm(-2) and merocyanine 540-mediated laser light treatment were studied. Isolated erythrocyte membranes (protein concentration of 1 mg/ml) were irradiated by Nd:YAG laser (532 nm, 30 mW) and then incubated in a medium with 2 mM ATP for 30 min. Activity of ATPase was determined colorimetrically by measuring the colored reaction product of liberated inorganic phosphate and malachite green at 640 nm. Contribution of Na(+)/K(+)-ATPase to overall phosphate production was determined using ouabain. A positive effect of green laser light on Na(+)/K(+)-ATPase activity was observed. The dependence of enzymatically liberated inorganic phosphate on light fluence showed a linear correlation (R(2)=0.96, P=0.0005) for all fluences applied (9.5-63.3 J.cm(-2)). On the other hand, MC 540-mediated phototreatment caused a suppression of enzyme activity.

Mitochondrial alterations induced by 532 nm laser irradiation.

Mitochondrial alterations were monitored after low power green laser (532 nm, 30 mW) irradiation in the case of whole cells (B-14) and isolated mitochondria (from Wistar rat heart). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) assay products were significantly higher (by 8%) in irradiated B-14 cells as compared to non-irradiated controls. Mitochondrial transmembrane potential of B-14 cells, measured by means of a fluorescent probe 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)), significantly increased (by 13%) after exposure to green laser irradiation. Another MTT assay was used for isolated mitochondria suspensions in order to examine the effect of green laser irradiation on stimulation of processes related to oxidative phosphorylation. It revealed 31.3%-increase in MTT assay products in irradiated mitochondria as compared to controls. Laser irradiation of isolated mitochondria suspension did not significantly change 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence anisotropy, indicating that mitochondrial membrane fluidity was not affected by laser light. Fluorescence emission spectra of irradiated as well as non-irradiated mitochondria suspensions showed fluorescence maximum at 635 nm, corresponding to emission of Protoporphyrin IX, which was significantly lower (by 20.7%) in irradiated sample.

The effect of green laser light irradiation on whole blood platelets.

BACKGROUND: Laser light irradiation is assumed to have biostimulating effect in various cell types. However, there is still a lack of information concerning response of blood platelets to laser light irradiation. METHODS: In our study we used flow cytometry to monitor the effect of a green Nd-YAG laser (532 nm, 30 mW) irradiation on platelet activation and the expression of activated GPIIbIIIa glycoprotein complex (fibrinogen receptor) of whole blood platelets stained with fluorolabelled monoclonal antibody PAC-1. Also the formation of platelet microparticles and aggregates in a population of whole blood platelets following such irradiation was evaluated. RESULTS: Effects of laser light on platelet activation and reactivity were significant over a wide range of applied energies (p<0.01). While low and medium laser light energies (18 and 54 J) increased platelet activation, the irradiation with a high-energy laser light (108 J) resulted in depressed platelet reactivity and attenuated platelet response to activators. In addition, laser light irradiation had significant influence on the formation of platelet microparticles in either resting (p<0.05) or ADP-activated (p<0.05) platelets, while no significant effect was observed in collagen-activated platelets. On the other hand, laser light irradiation significantly increased the formation of platelet aggregates both in resting (p<0.01) and agonists-activated (p<0.05) platelets. CONCLUSIONS: Our results clearly point that the laser light irradiation of blood platelets can trigger signal transduction, leading to platelet activation, as well as the gradual loss of natural platelet reactivity and platelets' ability to respond to activating agents.

Changes of compressibility of low density lipoproteins following copper mediated oxidation.

The methods of measuring the ultrasound velocity and density were used for study the adiabatic compressibility of low density lipoproteins (LDL) during their oxidation. We showed, that copper-mediated oxidation of LDL resulted in a decrease of apparent specific compressibility, phi(k)/beta0, of lipoproteins. The changes of ultrasound velocity and phi(k)/beta0 value started much earlier than the beginning of propagation phase corresponding to the fast increase in concentration of conjugated dienes, measured by absorption at 230 nm. It was assumed that the changes of compressibility could be in particularly due to increase in ordering of the phospholipids during reductive activation of Cu2+.

Impaired erythrocyte transmembrane potential in diabetes mellitus and its possible improvement by resorcylidene aminoguanidine.

Erythrocytes of diabetic patients have abnormal membrane properties. We examined in vitro transmembrane potential and the possible effect of resorcylidene aminoguanidine (RAG) on its modulation in erythrocytes of diabetic subjects. The transmembrane potential was assessed in RAG-treated and untreated erythrocytes, respectively, using a fluorescent dye (3,3'-dipropylthiadicarbocyanine iodide [DiSC3(5)]). We confirmed earlier findings that the transmembrane potential of diabetic erythrocytes is significantly increased compared with control (P < 0.01). The membrane hyperpolarization found in diabetic cells seems to be a result of oxidative stress present in diabetes mellitus. On one hand, the RAG treatment induced decrease in abnormal transmembrane potential values in diabetic erythrocytes (P < 0.01), presumably via its antioxidant and antiglycation activity. On the other hand, RAG moderately hyperpolarized the control erythrocytes (P < 0.05). We suggest that the drug-induced transient membrane expansion leads to an intracellular potassium loss and a subsequent change of the transmembrane potential. However, if controlled by an appropriate dosage, RAG can eliminate certain types of erythrocyte membrane damage induced by diabetes mellitus.

[Effect of vitamin C and E on nonenzymatic glycation and physico- chemical properties of isolated erythrocyte membranes in diabetic patients]. / Vplyv vitamínu C a E na neenzýmovú glykáciu a fyzikálno-chemické vlastnosti izolovaných membrán erytrocytov u diabetických pacientov.

Non-enzymatic glycation, accompanied by the formation of free radicals, represents a serious problem in diabetes mellitus. It is supposed to be the cause of the development of long-term diabetic complications. The aim of this work was to estimate the effect of treatment with vitamin C (1 g per day) and E (600 mg per day) on selected biochemical parameters as well as to determine the physicochemical state of erythrocyte membranes in diabetics. The paper also compares the physicochemical state of diabetic and control erythrocyte membranes. The changes in the values of glycaemia, glycated haemoglobin, and fructosamine were insignificant after three months of treatment. This points out that the doses used could be low or that the patient compliance was poor. An anionic fluorescent probe merocyanine 540 (MC540) was used to monitor possible changes in the physicochemical properties of isolated diabetic erythrocyte membranes. Significantly higher affinity of MC540 monomers to the membrane in diabetics treated with vitamin E was observed, which can be the result of the antioxidative effect of the vitamin (p < 0.02). A comparison of absorption spectra of MC540 in diabetic and control membranes revealed significant changes in the position of the bands and in their absorbances (p < 0.01 and less). They result from substantial alterations in the structure, surface charge, and the fluidity of erythrocyte membranes in diabetes mellitus. (Tab. 2, Fig. 3, Ref. 22.)

Decreased fluidity of isolated erythrocyte membranes in type 1 and type 2 diabetes. The effect of resorcylidene aminoguanidine.

Changes in the physico-chemical properties of erythrocyte membranes induced by nonenzymatic glycation as well as the possible prevention of their rise were studied. Using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH), fluorescence anisotropy values were determined in erythrocyte membranes isolated from type 1 and type 2 diabetic patients with and without complications. The mean anisotropy values for the groups of diabetic patients were significantly higher than those for the control group (p < 0.01). This indicated pathologically decreased fluidity in cell membranes in the diabetics regardless of the type of diabetes or the presence of complications. The fluorescence anisotropy positively correlated (p < 0.01) with clinical parameters, such as glycohaemoglobin and plasma cholesterol content, which are important for the monitoring of the compensation status of the diabetic patient. Our results support the suggestion that protein crosslinking and oxidative stress induced by nonenzymatic glycation contribute to changes in the physico-chemical properties of erythrocyte membranes. In vitro testing of a new potential drug resorcylidene aminoguanidine (RAG) showed its ability to increase significantly (p < 0.001), to various extent (p < 0.01), the fluidity of both diabetic and control erythrocyte membranes. Upon the administration of RAG, reduced fluorescence anisotropy values for the groups of diabetic patients approached the normal values obtained for the controls. This may play an important role in the improvement of impaired cell functions found in diabetes that are controlled by the cell membrane.