Cardiovagal regulation and transcutaneous pO2 in breast cancer patients - a pilot study.

Vagal activity in patients with metastatic or recurrent breast cancer can predict their survival and it can be altered by behavioral, pharmacological and surgical interventions. Tumor oxygenation is important in defining the cellular metabolic microenvironment of human malignancies, O2-depleted areas coincide with nutrient and energy deprivation and with a hostile metabolic microenvironment. In our work, we simultaneously measured two oxygen-sensitive parameters in breast cancer patients; blood oxygen saturation (SpO2) and trans-cutaneous O2 partial pressure (tcpO2) in breast tissue. Concurrently, 5-minute beat-to-beat heart rate recording was carried out in order to get heart rate variability (HRV) data from time-domain analyses, frequency-domain analyses and entropy and symbolic dynamic non-linear methods. We compared these parameters in patients newly diagnosed with breast cancer, in patients after therapy and in healthy controls. We found lower tcpO2 in patients with presence of malignant tumor compared to those post-treatment and/or without presence of malignancy. We also detected lower 2UV% (two unlike variations) and entropy in non-linear HRV analysis in all breast cancer patients and these parameters associated with parasympathetic activity did not return to the values comparable with healthy individuals after anti-cancer therapy, contrary to tcpO2. Our findings show that breast tissue tcpO2 can recover after the anti-cancer treatment, but complex heart rate control and cardio-vagal regulation remain impaired. This supports the idea that cancer patients and survivors might benefit from non-pharmacological interventions aimed at enhancing vagal activity, such as HRV biofeedback or Yoga.

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.

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.

Lactase haplotype diversity in the Old World.

Lactase persistence, the genetic trait in which intestinal lactase activity persists at childhood levels into adulthood, varies in frequency in different human populations, being most frequent in northern Europeans and certain African and Arabian nomadic tribes, who have a history of drinking fresh milk. Selection is likely to have played an important role in establishing these different frequencies since the development of agricultural pastoralism approximately 9,000 years ago. We have previously shown that the element responsible for the lactase persistence/nonpersistence polymorphism in humans is cis-acting to the lactase gene and that lactase persistence is associated, in Europeans, with the most common 70-kb lactase haplotype, A. We report here a study of the 11-site haplotype in 1,338 chromosomes from 11 populations that differ in lactase persistence frequency. Our data show that haplotype diversity was generated both by point mutations and recombinations. The four globally common haplotypes (A, B, C, and U) are not closely related and have different distributions; the A haplotype is at high frequencies only in northern Europeans, where lactase persistence is common; and the U haplotype is virtually absent from Indo-European populations. Much more diversity is seen in sub-Saharan Africans than in non-Africans, consistent with an "Out of Africa" model for peopling of the Old World. Analysis of recent recombinant haplotypes by allele-specific PCR, along with deduction of the root haplotype from chimpanzee sequence, allowed construction of a haplotype network that assisted in evaluation of the relative roles of drift and selection in establishing the haplotype frequencies in the different populations. We suggest that genetic drift was important in shaping the general pattern of non-African haplotype diversity, with recent directional selection in northern Europeans for the haplotype associated with lactase persistence.

High frequency of alkaptonuria in Slovakia: evidence for the appearance of multiple mutations in HGO involving different mutational hot spots.

Alkaptonuria (AKU) is an autosomal recessive disorder caused by the deficiency of homogentisate 1,2 dioxygenase (HGO) activity. AKU shows a very low prevalence (1:100,000-250,000) in most ethnic groups. One notable exception is in Slovakia, where the incidence of AKU rises to 1:19,000. This high incidence is difficult to explain by a classical founder effect, because as many as 10 different AKU mutations have been identified in this relatively small country. We have determined the allelic associations of 11 HGO intragenic polymorphisms for 44 AKU chromosomes from 20 Slovak pedigrees. These data were compared to the HGO haplotype data available in our laboratory for >80 AKU chromosomes from different European and non-European countries. The results show that common European AKU chromosomes have had only a marginal contribution to the Slovak AKU gene pool. Six of the ten Slovak AKU mutations, including the prevalent G152fs, G161R, G270R, and P370fs mutations, most likely originated in Slovakia. Data available for 17 Slovak AKU pedigrees indicate that most of the AKU chromosomes have their origins in a single very small region in the Carpathian mountains, in the northwestern part of the country. Since all six Slovak AKU mutations are associated with HGO mutational hot spots, we suggest that an increased mutation rate at the HGO gene is responsible for the clustering of AKU mutations in such a small geographical region.