A clinical evaluation of the TK 210 ELISA in sera from breast cancer patients demonstrates high sensitivity and specificity in all stages of disease.

Thymidine kinase (TK1) is an enzyme involved in DNA synthesis that leaks into the blood as a result of high cell turnover, particularly in the case of cancer. Serum TK1 activity has been used for prognosis and monitoring of leukemia and lymphoma patients for many years. Here, we describe the first clinical results with the newly developed TK 210 ELISA from AroCell AB. Sera from 124 breast cancer patients with known TNM classification along with sera from 53 healthy females were analyzed by TK 210 ELISA for TK1 protein and TK1 activity levels by the 3[H]-deoxythymidine (dThd) phosphorylation assay. The limit of detection for the TK 210 ELISA was 0.17 ng/ml, and 60 % of the sera from female blood donors were below this value. The median TK1 levels found in sera from breast cancer patients with T1 to T4 stage disease were 0.31, 0.46, 0.47, and 0.55 ng/ml, and these levels significantly differed from healthy controls. The median values of the biomarker CA 15-3 were also increased in patient sera from T1 to T4 patients (16, 34, 36, 40 U/ml, respectively). TK 210 ELISA showed significantly higher sensitivity for the T1 and T2 breast cancer patients compared to the TK activity assay. The combination of the TK1 ELISA and CA 15-3 biomarkers demonstrated a significant increase in sensitivity up to 15 % compared to each marker alone. This evaluation of the TK 210 ELISA strongly suggests that it can provide independent and complementary information for patients with breast cancer.

Discovery of phosphatidylcholines and sphingomyelins as biomarkers for ovarian endometriosis.

BACKGROUND: Current non-invasive diagnostic methods for endometriosis lack sensitivity and specificity. In search for new diagnostic biomarkers for ovarian endometriosis, we used a hypothesis-generating targeted metabolomics approach. METHODS: In a case-control study, we collected plasma of study participants and analysed their metabolic profiles. We selected a group of 40 patients with ovarian endometriosis who underwent laparoscopic surgery and a control group of 52 healthy women who underwent sterilization at the University Clinical Centre Ljubljana, Slovenia. Over 140 targeted analytes included glycerophospholipids, sphingolipids and acylcarnitines. The analytes were quantified by electrospray ionization tandem mass spectrometry. For assessing the strength of association between the metabolite or metabolite ratios and the disease, we used crude and adjusted odds ratios. A stepwise logistic regression procedure was used for selecting the best combination of biomarkers. RESULTS: Eight lipid metabolites were identified as endometriosis-associated biomarkers due to elevated levels in patients compared with controls. A model containing hydroxysphingomyelin SMOH C16:1 and the ratio between phosphatidylcholine PCaa C36:2 to ether-phospholipid PCae C34:2, adjusted for the effect of age and the BMI, resulted in a sensitivity of 90.0%, a specificity of 84.3% and a ratio of the positive likelihood ratio to the negative likelihood ratio of 48.3. CONCLUSIONS: Our results suggest that endometriosis is associated with elevated levels of sphingomyelins and phosphatidylcholines, which might contribute to the suppression of apoptosis and affect lipid-associated signalling pathways. Our findings suggest novel potential routes for therapy by specifically blocking highly up-regulated isoforms of phosphpolipase A2 and lysophosphatidylcholine acyltransferase 4.

Biomarkers of low-level mercury exposure through fish consumption in pregnant and lactating Slovenian women.

In order to assess the mercury exposure of pregnant and lactating women in Slovenia, levels of total mercury (THg) and methylmercury (MeHg) were determined in hair, cord blood and breast milk. In addition, the frequency of fish consumption was estimated, because fish is generally the main pathway for human exposure to MeHg. Hair samples were collected from 574 women participating in this study, while cord blood and breast milk samples were collected from 446 and 284 women, respectively. As expected, the levels of THg in hair (median (Med)=297 ng/g, 10th percentile (P10)=73 ng/g, 90th percentile (P90)=781 ng/g), cord blood (Med=1.5 ng/g, P10=0.5 ng/g, P90=4.2 ng/g) and breast milk (Med=0.2 ng/g, P10=0.06 ng/g, P90=0.6 ng/g) were low, due to low consumption of fish (X=25 g/day). A significant linear correlation was found between levels of lnTHg in hair and lnTHg in cord blood (r=0.87, 95% confidence interval (CI): 0.84-0.89), between levels of lnTHg in hair and lnMeHg in cord blood (r=0.94, 95% CI: 0.90-0.96) and between lnTHg levels in cord blood and lnTHg levels in breast milk (r=0.36, 95% CI: 0.25-0.47). Spearman's rank correlations between the frequency of fish consumption and THg in hair (rs=0.35, 95% CI: 0.28-0.42), and between the frequency of fish consumption and THg in cord blood (rs=0.43, 95% CI: 0.36-0.51) or MeHg in cord blood (rs=0.31, 95% CI: 0.06-0.52) were weak. This could be due to the approximate information on fish consumption obtained from the questionnaires, the high variability of MeHg concentrations in fish and a relatively high proportion of inorganic mercury in the biomarkers which originates from sources other than fish. In conclusion, THg levels in cord blood, THg levels in hair and MeHg levels in cord blood are suitable biomarkers of low-level Hg exposure through fish consumption. Compared to cord blood, hair samples are easy to collect, store and analyse.

Polymorphisms in the promoter region of the basic fibroblast growth factor gene are not associated with myocardial infarction in a Slovene population with type 2 diabetes.

Basic fibroblast growth factor (bFGF) is a multifunctional growth factor that may play a significant role in atherosclerotic vascular complications in patients with type 2 diabetes. This study was designed to investigate the association between genetic polymorphisms (-553 T/A, -834 T/A and -921 C/G) in the promoter region of the bFGF gene and myocardial infarction (MI) in 443 patients with type 2 diabetes (149 with MI and 294 with no history of coronary artery disease). The -553 T/A, -834 T/A and -921 C/G polymorphisms of the bFGF gene were found not to be risk factors for MI in patients with type 2 diabetes. The impact of bFGF gene polymorphisms on serum bFGF levels was also investigated and significantly higher serum levels of bFGF were demonstrated in diabetes patients with the TA genotype of the -553 T/A polymorphism compared with diabetes patients with the TT wild type genotype (9.0 +/- 5.6 ng/l versus 3.0 +/- 1.9 ng/l, respectively). Thus, the tested bFGF gene polymorphisms cannot be used as genetic markers for MI in diabetic Caucasians.

S100B protein in benzodiazepine overdose.

BACKGROUND: Severe benzodiazepine overdose can result in coma and respiratory depression that might cause brain hypoxia, necrosis and delayed post-anoxic leucoencephalopathy with permanent neurological sequelae. The aim of this study was to assess the possible role of S100B, a structural protein of astroglial cells, as a biochemical marker of brain injury in acute benzodiazepine overdose. METHODS: Serum S100B determination was performed in 38 consecutive patients admitted to the emergency department (ED) in Ljubljana with benzodiazepine overdose. The level of consciousness and respiratory insufficiency on the scene were assessed by responsiveness to a verbal stimulus and pulse oximetry. Blood samples were taken immediately after arrival at the ED and S100B concentrations were measured with a commercial immunoluminometric assay. 20 healthy sex- and age-matched volunteers formed a control group. RESULTS: There were significant differences in S100B levels between the control group and the patients with benzodiazepine overdose according to their responsiveness to a verbal stimulus. Post hoc test results showed that S100B levels in patients with benzodiazepine overdose who were unresponsive to a verbal stimulus were significantly higher than those in patients responsive to a verbal stimulus (median 0.31 vs 0.11 microg/l; p = 0.001). Both groups of patients with benzodiazepine overdose had significantly higher S100B levels than the control group (median 0.07 microg/; both p = 0.001). Arterial oxygen saturation of patients with benzodiazepine overdose unresponsive to a verbal stimulus was significantly lower than in patients responsive to a verbal stimulus (median 83% vs 94%; p = 0.001). There was no significant difference in the systolic blood pressure of patients with benzodiazepine overdose responsive or unresponsive to a verbal stimulus. CONCLUSION: Raised levels of S100B protein are associated with depressed levels of consciousness and respiratory insufficiency in patients with benzodiazepine overdose.

Use of lysophosphatidic acid in the management of benign and malignant ovarian tumors.

OBJECTIVE: To establish whether LPA determination improves the differentiation of benign from malignant ovarian tumors. METHODS: Total LPA and LPA species in the serum were determined using a novel method in 142 patients with ultrasound (US) suspecious ovarian tumors and in 78 healthy women. All women underwent determination of CA125 in the serum, a vaginal US examination and morphology scoring of the tumor. RESULTS: The levels of total LPA and its species in women with ovarian tumors were significantly higher from those in healthy women (p < 0.001). No significant difference was found in the levels of total LPA or any of its species between the women with benign and those with malignant ovarian tumors. CONCLUSIONS: Determination of serum LPA would be an appropriate test for ovarian tumor presence, especially in women of reproductive age. The method however does not differentiate benign from malignant ovarian tumors.

Metabolic and cardiovascular changes in women with polycystic ovary syndrome.

OBJECTIVE: To analyze the relationship between QTc interval and cardiovascular risk factors in women with polycystic ovary syndrome (PCOS). METHODS: Study group included 119 PCOS women (age: 32.2+/-5.2 years) and the control group 64 age-matched healthy women; they all underwent QT interval measurement, and plasma levels of high-sensitivity CRP (hsCRP), endothelin-1 (ET1), insulin, and testosterone determinations. RESULTS: In PCOS women hsCRP (2.35+/-2.14 mg/L vs. 1.01+/-1.28 mg/L; P=0.04), ET1 (23.6+/-10.3 ng/L vs. 7.7+/-15.9 ng/L; P=0.01), and insulin (16.5+/-7.8 mIU/L vs. 11.8+/-10.7 mIU/L; P=0.03) levels were significantly higher, and QTc interval significantly shorter than in controls (401+/-61 ms vs. 467+/-61 ms; P=0.007). In 67 (56%) PCOS patients with a short QTc interval (<400 ms), plasma testosterone levels were significantly higher than in PCOS women with normal QTc interval (2.3+/-2.1 nmol/L vs. 1.4+/-1.7 nmol/L; P=0.02). CONCLUSIONS: In patients with polycystic ovary syndrome increased testosterone levels may attenuate the effects of coronary risk factors.

GH-binding protein is related to bone mineral density in non-obese androgenized women.

GH and IGF system components are important regulators of bone formation and at the same time pathogenetic factors in functional hyperandrogenism (FH) in lean females. We studied the relationships between bone mineral density (BMD) and serum concentrations of GH, GH-related parameters, androgens and estrogen, in 18 non-obese women of reproductive age with functional hyperandrogenism compared to a group of 10 healthy eumenorrheic age- and weight-matched women. In androgenized women, a significant positive correlation was found between BMD and GH-binding protein (GHBP), whereas BMD did not correlate to GH or other related parameters. It is suggested that higher tissue GH receptor responsiveness in non-obese androgenized women may contribute to their higher BMD.

Relevance of meat fat content and fruit and vegetable intake for the oxidative status of pigs.

BACKGROUND: The aim of the present study was to evaluate the effect of substituting lean meat with fat meat on oxidative stress in a diet with or without fruit and vegetables. METHODS: Thirty-two pigs were divided into groups and fed isocaloric daily rations: LM+FV (balanced diet with lean meat and fruit and vegetables); FM+FV (as LM+FV, but lean meat was substituted with fat meat); LM-FV (as LM+FV, but without fruit and vegetables), and FM-FV (as FM+FV, but without fruit and vegetables). Oxidative stress was evaluated by measuring the 24-hour urine malondialdehyde excretion rate, the degree of leukocyte nuclear DNA damage, the concentration of tocopherols in blood plasma, erythrocyte glutathione peroxidase activity and the total antioxidant status of plasma. RESULTS: The substitution of lean meat with fat meat modestly increased the rate of leukocyte DNA damage only in the diet with fruit and vegetables but had no effect in the group deprived of fruit and vegetables. Regardless of the fruit and vegetable content of the diet, the substitution of lean meat with fat meat did not affect any other parameters measured. In comparison to both fruit- and vegetable-containing diets, the deprivation of fruits and vegetables in the LM-FV and FM-FV groups significantly increased the rate of leukocyte DNA damage and reduced the plasma alpha-tocopherol level (significant only for FM+FV). CONCLUSION: The substitution of fat meat with lean meat in a diet with or without fruit and vegetables has only a marginal or no effect on oxidative stress. But fruit and vegetable exclusion markedly increased the level of oxidative stress.

GH-IGF-I axis in non-obese women with functional hyperandrogenism.

The study was conducted to assess the GH-IGF-I axis in non-obese women with functional hyperandrogenism (FH). Eighteen FH women aged 18-35 yr with a body weight within 20% of ideal body weight and 10 weight-matched controls were included in the study. Basal serum GH, GH-binding protein (GHBP), IGF-I, IGF-binding protein-3 (IGFBP-3) levels were determined as well as GH levels during GHRH stimulation. In addition, basal serum androgens [free T (FT), delta4 and DHEAS], insulin and glucose levels were determined. The group of non-obese patients with FH differed from controls in GHBP (1.21+/-0.37 vs 0.93+/-0.25 nmol/l; p<0.05) and androgen levels (FT: 8.0+/-3.2 vs 1.9+/-1.2 pmol/l, p<0.001; delta4: 10.5+/-3.2 vs 5.9+/-2.1 nmol/l, p<0.001; DHEAS: 9.3+/-3.0 vs 5.1+/-1.8 micromol/l, p<0.001). GH (r=0.365; p<0.05) and IGF-I (r=0.508, p<0.01) serum levels were significantly correlated to serum DHEAS levels in a combined group of patients and controls. Our results support the suggestion that the GH-IGF-I axis plays an important role in the evolution of hormonal and metabolic derangement in non-obese FH women.

Autonomic nervous system function in patients with acute brainstem stroke.

Dysfunction of the autonomic nervous system (ANS) often complicates the clinical course in patients with acute stroke. The studies of the function of ANS in patients with brainstem stroke are scarce. The purpose of our study was to evaluate the function of ANS in the early period after acute brainstem stroke and to find out whether the location of stroke in brainstem influences the function of ANS. We studied heart rate variability (HRV) and plasma levels of catecholamines in 14 eligible patients with medullary (6 patients) and non-medullary (8 patients) brainstem stroke during the first 6 weeks after stroke. Integrals over the low- (LFB; 0.04-0.15 Hz) and high-frequency bands (HFB; 0.15-0.40 Hz) of power spectra were calculated. When compared to controls, the integrals over HFB in the hyperacute (p < 0.001) and over LFB in the hyperacute (p < 0.005) and in the acute (p < 0.05) phases were significantly smaller in patients with medullary strokes. Integrals over LFB and HFB in patients with non-medullary strokes did not differ significantly from controls, regardless of the phase of the disease. Plasma levels of epinephrine in patients with non-medullary stroke in the hyperacute and in the acute phases were significantly higher than in controls (p < 0.005). We conclude that there is transient dysfunction of ANS in patients with acute medullary stroke, in contrast to those with non-medullary brainstem stroke.

Effect of epidural and intravenous clonidine on the neuro-endocrine and immune stress response in patients undergoing lung surgery.

The effects of intravenous and epidural clonidine, 4 microg kg-1, combined with epidural morphine, 40 microg kg-1, on the neuro-endocrine and immune stress responses to thoracic surgery are reported. A control group received only epidural morphine. Anaesthesia was induced and maintained with propofol. Catecholamines, vasopressin, cortisol, beta-endorphin concentrations and leucocyte counts were measured before drug administration, immediately after intubation of the trachea, after thoracotomy and at the end of surgery. Catecholamines did not change in any of the groups. The other stress hormones increased during surgery, the pattern being similar in the three groups. Total leucocyte and neutrophil counts were increased in all groups at the end of surgery, but the increase was least in the epidural clonidine group. The number of lymphocytes was reduced at the end of surgery in the epidural and intravenous group, compared with the control group in which the number of lymphocytes did not change. The effects are more pronounced with epidural than with intravenous administration. We conclude that clonidine can modulate the immune stress response to thoracic surgery.

Insulin-like growth factor binding protein-3 (IGFBP-3) serum concentrations and ovarian responsiveness in in-vitro fertilization.

The growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis seems to play an important role in ovarian responsiveness. Recently IGF binding protein-3 (IGFBP-3) serum concentrations have been reported to be a good marker of GH/IGF-I axis activity. In view of this finding, we measured IGFBP-3 serum concentrations in 29 women undergoing in-vitro fertilization. We found a significant correlation among IGFBP-3 serum concentrations and markers of ovarian stimulation including efficacy index, serum oestradiol concentrations and the number of follicles on the day of human chorionic gonadotrophin (HCG) administration. The results of our study add additional evidence to the importance of the GH/IGF-I system in regulating ovarian responsiveness to gonadotrophin stimulation.

Androstanediol glucuronide in patients with pancreatic cancer and in those with chronic pancreatitis.

The differential diagnosis between pancreatic cancer and chronic pancreatitis is extremely difficult. Beside CA19-9 level determinations, many tests have been tried with the aim to facilitate this distinction. Serum androgen levels have been used for this purpose. To further explore the value of androgen markers in differentiating pancreatic cancer from chronic pancreatitis we determined the serum levels of androstanediol glucuronide and of androgens in the two groups of patients and compared them with CA19-9 levels. A total of 25 males were entered into the study. Of these, 13 patients had pancreatic cancer and 12 chronic pancreatitis. They were comparable as to their body weight and age. Patients with pancreatic cancer had significantly lower serum testosterone, dihydrotestosterone and androstanediol glucuronide levels, but not testosterone/dihydrotestosterone ratios when compared to patients with chronic pancreatitis. Only androstanediol glucuronide and dihydrotestosterone serum concentrations had such a small overlap between the two groups that could be used for differentiation, their sensitivity and specificity being comparable to those of CA19-9 levels. The present study has shown for the first time that serum androstanediol glucuronide levels in male patients with pancreatic cancer are significantly lower than in those patients with chronic pancreatitis. Furthermore, the sensitivity and specificity of serum andorstanediol glucuronide levels which can be used to differentiate between pancreatic cancer and chronic pancreatitis are comparable to those of CA19-9.

Effect of elevated plasma norepinephrine on electrocardiographic changes in subarachnoid hemorrhage.

We compared electrocardiographic abnormalities and plasma norepinephrine concentrations in 40 patients with subarachnoid hemorrhage within the first 24 hours, at 72 hours, and after 1 week. In 20 patients with high plasma norepinephrine concentrations within the first 24 hours, sinus tachycardia (p less than 0.02) and negative T waves (p less than 0.01) were more frequent than in the 20 patients with normal plasma norepinephrine concentrations. After 72 hours, only sinus tachycardia (p less than 0.03) was found with increased frequency in the 26 patients with high plasma norepinephrine concentrations. Although 24 patients had high plasma norepinephrine concentrations after 1 week, we found no differences in the frequency of electrocardiographic abnormalities compared to patients with normal plasma norepinephrine. However, QTc prolongation, U waves, ST depression, and arrhythmias were found with similar frequency in patients with both high and normal plasma norepinephrine concentrations. We conclude that, with the exception of sinus tachycardia and negative T waves, electrocardiographic changes in patients with subarachnoid hemorrhage do not depend on elevated plasma norepinephrine concentrations.

Salivary free testosterone in hirsutism.

A sensitive, specific and accurate direct radioimmunoassay of testosterone in human saliva is described. A single salivary testosterone result is shown to be of greater diagnostic use in hirsutism than any of the currently used serum androgen assays. Thus, of 50 hirsute patients, salivary testosterone (Sa-T) was elevated in 34 patients, sex hormone-binding globulin (SHBG) was decreased in 30 women, serum testosterone (S-T) elevated in 13, dehydroepiandrosterone sulphate (DHEA-S) was elevated in 14, and androstenedione in three of the investigated group.