Jan Pachecka

Serum cortisol concentration in patients with major depression after treatment with fluoxetine

Hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis and elevated cortisol levels is characteristic of the pathophysiology of major depressive disorder (MDD). The aim of this study was to determine whether increased plasma cortisol levels appear in patients with major depression and if effective antidepressant treatment by fluoxetine leads to regulation of cortisol level. This aim was realized by describing and validation of methods of determining fluoxetine and cortisol in serum and searching for correlation between their concentrations in patients with endogenous depression, the therapeutic effect as assessed in Hamilton Depression Rating Scale (HDRS), age and sex of patients. Plasma cortisol and fluoxetine levels were measured using high performance liquid chromatography (HPLC) methods with applying Shimadzu chromatograph with UV detection. Plasma cortisol and fluoxetine levels were measured at time zero (before therapy) and after 6h, 24h, 2, 4, 6 and 8 weeks of fluoxetine administration in patients with major depression qualified for therapeutic drug monitoring (TDM). The study included 21 patients (14 women, 7 men; mean age 29-75 years) and 24 healthy comparison subjects. The patients had a mean score on the 21-item HDRS. As the effect of fluoxetine administration the decrease of the level of cortisol was observed in patients who responded to the therapy (the reduction of points in HDRS scale in at least 50%). The validation parameters of HPLC method of fluoxetine and cortisol determination indicate the possibility of applying them for determination of both: the level of concentration of the drug in therapeutic drug monitoring and the level of cortisol in serum of patients with endogenous depression.

Simultaneous determination of eight estrogens and their metabolites in serum using liquid chromatography with electrochemical detection.

The liquid chromatography (LC) with electrochemical detection allows to determine, evaluate and validate the level of estrogens and their metabolites in serum. The method is fast and sensitive, and the hormones can be determined simultaneously, from one serum sample. The proposed method was successfully applied to the determination of following estrogens: estrone (E(1)), 17beta-estradiol (E(2)), estriol (E(3)) and following catecholestrogens: 2-hydroxyestradiol (2-OHE(2)), 4-hydroxyestradiol (4-OHE(2)), 4-hydroxyestrone (4-OHE(1)), 2-methoxyestradiol (2-MOE(2)) and 2-methoxyestrone (2-MOE(1)). The method of LC with electrochemical detection (LC/EC) was applied for the determination of catecholestrogens in serum sample taken from pregnant women. Estrogens and catecholestrogens were extracted from 1 mL of serum by applying diethyl ether under the specified conditions. The parameters of the procedure included using a specific mobile phase, applying the column Symmetry C18 (5 microm, 3.0 mm x 150 mm), equipping the applied electrochemical detector with the working glassy-carbon electrode, as well as applying the reference electrode Ag/AgCl. The calibration studies on this study were performed, and a good analytical performance for E(1), E(2), E(3), 2-OHE(2), 4-OHE(2), 4-OHE(1), 2-MOE(2) and 2-MOE(1) was attained, along with low limits of detection (LOD of 0.18-0.30 ng/mL), satisfactory limit of quantification (LOQ of 0.23-0.92 ng/mL) and excellent linear dynamic range (0.6-8.0 ng/mL). In conclusion, the presented methodology is the sensitive method of the simultaneous measurement of eight estrogens and their metabolites from one sample of blood and it might be clinically applied for testing serum of pregnant women, as well as for further studies on estrogens and their metabolites.

Serum cortisol concentration in patients with major depression after treatment with clomipramine.

Hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis and elevated cortisol (CORT) levels are characteristics of the pathophysiology of major depressive disorder. The aim of this study was to determine whether increased plasma CORT levels appear in patients with major depression and if effective antidepressant treatment by clomipramine (CLO) leads to regulation of CORT level. Plasma CORT levels were measured using high performance liquid chromatography (HPLC) methods in patients with major depression at time zero (before therapy) and after 3 h, 24 h, 4, 6 and 8 weeks of CLO administration. The study included 17 patients (12 women, 5 men; mean age 54.5 years, SD =12.3) and 21 healthy comparison subjects. The patients had a mean score on the 21-item Hamilton Depression Rating Scale (HDRS) of 26.8 (range 22-35). Eight of the patients with major depression recruited for the study showed a 46% increase in CORT concentration compared to the established standard. In 13 patients treated with CLO, serum CLO levels reached a therapeutic range. In recovered depressed patients, antidepressant treatment significantly reduced HDRS scores from the 6th week of treatment. A drop in plasma CORT levels in recovered depressed subjects occurred 0 to 6 weeks after CLO treatment (n = 5, p < 0.046). However, neither subject group exhibited any definitive markers of CORT secretion. In the population studied, patients had distinct profiles of HPA axis dysregulation. Finding a linear correlation between lower CORT secretion and therapeutic plasma CLO levels is the first aim of monitored therapy and may be important for understanding the pathophysiology of major depressive disorder.

Serum alpha-1-antitrypsin concentration during normal and diabetic pregnancy

OBJECTIVES The aim of this study was a comparison of serum alpha-1-antitrypsin (AAT) concentration in the course of normal and diabetic pregnancy. METHODS Serum AAT concentration was determined on NOR-Partigen plates (Behring Diagnostics GmbH, Marburg). The studied material included healthy women without pregnancy (n=14), healthy pregnant women in the first trimester (n=12), second trimester (n=15), third trimester (n=15), and 16 pregnant women with type-1 diabetes mellitus studied prospectively in successive stages of pregnancy. RESULTS In the first trimester of normal pregnancy, a significant increase of serum AAT concentration was observed in comparison with healthy women without pregnancy (P<0.01). In all stages of pregnancy with type-1 diabetes mellitus, a higher increase of AAT concentration was found as compared with healthy pregnant women (P<0.0001), especially in third trimester. There was no correlation shown between concentration of AAT and fructosamine in the serum of healthy and diabetic pregnant women (P>0.05). CONCLUSIONS During normal and diabetic pregnancy, an increase of serum AAT concentration occurred with the regression lines exhibiting a different slopes. The highest AAT concentration was observed in third trimester of diabetic pregnancy. Increase in concentration of AAT in the serum of pregnant women with diabetes does not depend on the value of glycaemic control.

Antigenic and functional levels of alpha-1-antitrypsin in serum during normal and diabetic pregnancy

OBJECTIVES Serum alpha-1-antitrypsin (AAT) concentration may not be representative of the functional capacity of this inhibitor. The aim of this study was to determine the antigenic and functional serum levels of AAT during normal and diabetic pregnancy. METHODS Serum AAT concentration was measured on NOR-Partigen plates (Dade Behring). Trypsin inhibitory capacity (TIC) in the serum was determined with N-benzoyl-DL-arginine-p-nitroaniline (BAPNA, Sigma) as substrate. The examined material included pregnant women with type 1 diabetes mellitus (n=16) studied prospectively in successive stages of pregnancy, healthy pregnant women in the first trimester (n=12), second trimester (n=15), third trimester (n=15) and healthy non-pregnant women (n=14). RESULTS Serum concentration of AAT in all consecutive phases of diabetic pregnancy is higher as compared to normal pregnancy (P<0.0001). Serum TIC is significantly lower in the first and third trimesters in diabetic pregnancy (P<0.05, 0.001, respectively). Specific activity of serum AAT (mg of trypsin inhibited by 1mg of AAT) does not change between subsequent trimesters both in normal and diabetic pregnancy and in diabetic pregnancy is two times lower as compared to normal pregnancy. CONCLUSION In spite of the higher level of AAT in the serum in diabetic pregnancy, the ability of this inhibitor to inhibit trypsin is two times lower as compared to normal pregnancy.

Urinary proteins, N‐acetyl‐β‐D‐glucosaminidase activity and estimated glomerular filtration rate in hypertensive patients with normoalbuminuria and microalbuminuria

Background:  The aim of the study was to assess novel candidate markers measured in the urine of normoalbuminuric and microalbuminuric patients (the urinary albumin‐to‐creatinine ratio < 30 mg/mmol) with essential hypertension to be used for early detection and assessment of progressive deterioration in renal function.

Alpha-1-antitrypsin and IgA in serial meconium and faeces of healthy breast-fed newborns.

Background: Meconium is a series of layers formed in the foetal intestine from the 12th week of gestation. High content of meconial alpha-1-antitrypsin (AAT), decreasing within the first several days of extrauterine life appears to reflect the meconium clearance of the gut. At birth, IgA is not present in the meconium and breast-fed infants receive this antibody postnatally with human milk. The aim of the study was to determine changes in AAT concentrations, functional activity of that inhibitor expressed as trypsin inhibitory capacity (TIC) and IgA concentration in serial meconium and faeces, as endogenous biochemical markers discriminating between faeces portions formed in intrauterine and extrauterine life periods of healthy breast-fed newborns. Methods: A group of 24 healthy breast-fed newborns delivered by spontaneous labour were studied prospectively during the first 4 days of postnatal life. AAT and IgA concentrations in the newborn‘s meconial and faecal samples and IgA concentration in mother’s milk samples taken on the third day after delivery, were determined by radial immunodiffusion. TIC was assessed using BAPNA (N-benzoyl-DL-arginine-p-nitroanilide). Results: The medians (range) of AAT concentrations in milligrams per gram of dry meconium or faeces were: 68.8(29.2–138.4) (day 1), 56.9 (30.8–112.8) (day 2), 26.2 (6.8–80.7) (day 3), and 6.6 (1.4–27.1) (day 4). The medians (range) of TIC in milligrams of trypsin/g dry mass of meconium or faeces were: 0.76 (0.33–1.79) (day 1), 0.44 (0.17–1.08) (day 2), 0.16 (0.03–0.56) (day 3), and 0.03 (0–0.11) (day 4). The median (range) of IgA concentration in mothers’ milk was 715 mg/dl (420–890). IgA was absent in meconium portions from the first day of life while on the successive days the medians (range) of IgA concentration in mg/g dry mass of meconium and faeces were as follows: 0 (0–2.90) (day 2), 2.50 (1.10–9.60) (day 3), 7.05 (4.10–30.60) (day 4). On day 4 of extrauterine life a negative correlation was found between AAT and IgA concentrations in faeces of the newborns (r = –0.46) and a positive correlation was seen between IgA concentrations in faeces and milk (r = 0.93). Conclusions: Analyses of the systematic decrease in AAT and increase of IgA concentration in serial portions of meconium and faeces over the first days of extrauterine life of breast-fed newborns can date newborn’s faeces portions formed during intrauterine and extrauterine maturation. AAT deposited in foetal intestine is an active antiprotease.

Trypsin and antitrypsin activities and protein concentration in serial meconium and feces of healthy newborns.

Background. Meconium has the potential for being the matrix on which markers of fetal exposure to physiologic and non-physiologic agents during intrauterine life can be analyzed. The aim of this study was to compare trypsin and antitrypsin activities and protein concentration during intra- and extrauterine human development based on an assessment of these parameters in serial meconium and first feces of healthy, term newborns during the first four days of life. Methods. One hundred and eighteen mature, term newborns were studied. Single portions of meconium or feces were taken prospectively from day 1 to day 4. In ten newborns each meconium and feces passed by the infant from birth up to the fourth day after delivery was collected individually. Trypsin activity was measured using L-TAPA (N-α-tosyl-l-arginine-p-nitroanilide) as substrate, trypsin inhibitory capacity (TIC) with N-α-benzoyl-dl-arginine-p-nitroanilide (BAPNA) as substrate, and protein concentration by the method of Lowry et al. Results. Meconium and feces of healthy newborns demonstrated a decrease in TIC during the first two days of life and an increase in trypsin activity over the first three successive days of life. No correlation was observed between the variability of both parameters. During the first three days of a newborns life, significant correlation was observed between TIC and protein concentration in meconium and first feces. In the course of proteolytic activity changes in successive portions of meconium and feces, a transient peak occurs on the 2nd–4th day of life. Conclusions. The gradual decrease in protease inhibitor activity and low trypsin activity in successive portions of meconium from the first two days of extrauterine life may provide a retrograde chronological depiction of the course of the second and third trimesters of intrauterine life. Serial collection of feces over 2–4 days may provide a reflection of the dynamics of the postnatal increase in the newborns pancreatic exocrine functions.

Comparison of urinary excretion of albumin and alpha-1-antitrypsin in patients with arterial hypertension

The aim of the presented pilot study was to compare urinary excretion of two plasma proteins similar in molecular mass and isoelectric point: albumin and alpha-1-antitrypsin (AAT) in patients with different forms of arterial hypertension and in healthy subjects. The 24-h urinary excretion of albumin and AAT were assessed in 52 patients, 29 with essential hypertension and 23 with secondary hypertension, caused by renovascular hypertension, adrenal phaeochromocytoma and obturative sleep apnoea syndrome. The concentrations of albumin and AAT were determined by rocket immunodiffusion. An increase of mean albumin and AAT urinary excretion was demonstrated, as compared to the control group, both in the patients with essential hypertension and with secondary hypertension. In 93.2% of the healthy subjects no AAT presence in urine was detected. No correlation was found between the excretion of albumin and AAT with urine.

Hepatic Mitochondrial Redox Potential in Patients with Liver Metastatic Cancers and Circulatory Insufficiency

Arterial ketone body ratio (AKBR), which reflects hepatic intramitochodrial redox potential, was measured in 20 patients with Carcinoma hepatis metastaticum and good circulatory condition (group A), and 16 patients with Carcinoma hepatis metastaticum and chronic cardiogenic circulatory insufficiency (group B). Total ketone body concentration (TKB) and arterial oxygen tension (PaO2) was simultaneously determined. We have stated that AKBR values in both groups of patients were decreased below the normal level. AKBR values in group B were significantly lower than in group A. At the same time TKB values in both groups were statistically equal and significantly increased above the normal level. The levels of arterial oxygen tension (PaO2) in group A were physiologically high, whereas in group B were significantly decreased. Furthermore arterial oxygen tension of patients in group B correlated with AKBR values significantly. In group A we found statistically significant negative correlation between TKB and AKBR values. Our study indicate that the main mechanism which may explain the decrease of intrahepatic mitochondrial redox potential in patients with liver metastatic cancers and good circulatory condition, is the enhanced beta-oxidation of fatty acids, when the efficiency of NAD+ to NADH reduction in beta-oxidation pathway and tricarboxylic acid cycle is higher than re-oxidation of NADH to NAD+ in the oxidative phosphorylation. In patients with coexisting chronic cardiogenic circulatory insufficiency deprivation of blood oxygen supply initiate the irreversible dysfunction of oxidative phosphorylation.