Apolipoprotein E phenotypes and genotypes as determined by polymerase chain reaction using allele-specific oligonucleotide probes and the amplification refractory mutation system in children with insulin-dependent diabetes mellitus.

The frequency of apolipoprotein E (apo E) phenotypes and genotypes due to allelic variation at amino acids 112 and 158 was analysed in 50 children with type I diabetes. Phenotypes were determined by isoelectric focusing and genotypes by the technique of polymerase chain reaction using allele-specific oligonucleotide probes (PCR/ASO) and the amplification refractory mutation system (ARMS). Discrepancies between phenotypes and genotypes as assigned by PCR/ASO were observed in 12 (24%) cases and by ARMS in eight (16%) cases. Results revealed the apo E3/3 genotype, as assigned by ARMS, to be the most frequent one (70%), followed by apo E3/4 in 16%, apo E2/2 in 2%, apo E2/3 in 8%, apo E2/4 in 2% and apo E4/4 in 2% of the cases. Apo E3/4 genotype and phenotype were more frequently present in the children with type I diabetes as compared with the diabetic adults previously reported on.

Products of advanced glycation in patients with type 2 diabetes and vascular disease.

BACKGROUND: Non-enzymatic glycation leading to advanced glycation endproduct (AGE) formation is thought to contribute to vascular pathology. In the present study, AGEs and anti-AGE antibodies in free and immune complex-bound form were assayed in the serum of diabetic (DMCAD) (n = 69) and nondiabetic (n = 78) patients with coronary artery disease (CAD) and in control subjects (n = 47) free from vascular disease. METHODS: A blocking enzyme-linked immunosorbent assay (ELISA) was used to test immunoreactivity against AGE epitope(s) and a competitive ELISA was used to measure total AGE content. RESULTS: Anti-AGE immunoreactivity was significantly higher in diabetic than in control subjects (P = 0.045). Although a wide range of anti-AGE antibody titres were observed in nondiabetic CAD patients, there was no significant difference from those of control subjects. Both diabetic and nondiabetic CAD patients had a higher concentration of circulating immune complexes containing the AGE moiety as antigen than did control subjects (DMCAD versus control, P = 0.041; CAD versus control, P = 0.047). Study patients showed a positive correlation between serum AGE and AGE-immune complexes (DM, r = 0.29, P = 0.014; CAD, r = 0.26, P = 0.019), whereas no such correlation was recorded in controls (r = 0.08, P = 0.89). CONCLUSION: To our knowledge, this is the first study demonstrating increased AGE-immune complexes in patients with CAD, either with or without diabetes, suggesting that AGE-immune complexes might be involved in the atherosclerotic process, either as the result of it or as part of the pathophysiologic process.

Treatment of acute cholecystitis. A comparison of open vs laparoscopic cholecystectomy.

BACKGROUND: In this study, the clinical results and cost-effectiveness of open vs laparoscopic cholecystectomy in the treatment of acute cholecystitis were compared. METHODS: Over a 5-year period (1994-98), 894 cholecystectomies were performed, 545 (60.96%) of them laparoscopically and 349 (39.04%) by the open method. The study included 209 patients with a clinical diagnosis of acute cholecystitis; 115 (55.02%) of them were operated on by the open method and 94 (44.98%) by the laparoscopic method. RESULTS: A comparison analysis revealed that the mean postoperative treatment period was 8.40 days after open and 4.38 days after laparoscopic cholecystectomy. In the group operated on by the open method, 106 patients received an antibiotic, a mean of 5.09 ampules and 3.2 tablets or suppositories of an analgesic, and 2.91 dressings per patient; whereas in the group submitted to the laparoscopic method, the comparable figures were 43, 3.13, 2.1, and 1.47, respectively. In 31 (26.96%) employed patients operated on by the open method, the mean absenteeism from work was 42 days; whereas in 31 (32.98%) of those operated on by the laparoscopic method, it was 17 days. The mean operating times for the procedures were 89 and 115 min for the open and laparoscopic methods, respectively. Two patients submitted to open cholecystectomy died within 30 days postoperatively. Wound infection was recorded in 10 (8.7%), prolonged biliary secretion in two, and cicatricial hernia in five (4.35%) patients. In the group submitted to laparoscopic cholecystectomy, there were no deaths; nine (9.57%) conversions were required; four patients had to be reoperated on, two of them for bile lobe hemorrhage and two for massive biliary secretion from the open cystic duct; herniation at the site of supraumbilical incision developed in three patients, and infection developed at the same site in two (2.13%) patients. The hospital cost was significantly higher in laparoscopic patients ($1181 vs $873) USD), as was the total cost of treatment for acute cholecystitis ($1430 vs $1316). However, the cost for sick leave and rehabilitation was significantly lower in laparoscopically treated patients ($486 vs $1199). CONCLUSIONS: Our comparison analysis of the results and cost-effectiveness of the surgical treatment of acute cholecystitis clearly pointed to the advantages of laparoscopic over open cholecystectomy-i.e., better clinical outcome and a more rapid resumption of daily activities. Hospital and total costs of treatment were on average higher in laparoscopic patients, except for the employed ones, where the lower sick leave cost translated into a significant reduction in total costs.

Evaluation of the Olympus AU 400 clinical chemistry analyzer.

The performance of the Olympus AU 400 clinical chemistry analyzer was evaluated according to the guidelines of the European Committee for Clinical Laboratory Standards. The following analytes were tested: glucose, urea, creatinine, calcium, AST, ALT, CK, LDH, ALP and amylase. The Olympus AU 400 was compared with the Olympus AU 800. Coefficients of correlation showed high correlation between the compared analyzers. Other performances (intra- and inter-assay variation, carry-over and interferences) of the analyzer were satisfactory.

Lack of association between burning mouth syndrome and hematinic deficiencies.

The aim of our investigation was to evaluate possible connection between burning mouth syndrome and hematinic deficiencies, a hypothesis previously reported in the literature with contradictory results. Serum levels of iron, vitamin B12, folic acid, calcium and magnesium were determined in 41 (aged 31-87 years, mean 68,7 yrs) patients with burning mouth syndrome and 35 matched controls (35-83, mean 63 yrs). Serum iron levels were determined according to Fairbanks and Klee. Levels of vitamin B12 and folic acid were determined on commercially available kits (Imx12 and Imx folate assay, Abbot Park lab, IL, USA) on Imx analyser. Calcium and magnesium levels were determined using atomic absorption spectrophotometry. No statistically significant differences in serum levels of iron, folic acid, calcium and magnesium were found between patients with burning mouth syndrome and controls. Statistically significant lowered vitamin B12 levels were found in patients with burning mouth syndrome. Our results suggest that serum deficiencies of iron, folic acid, calcium and magnesium are not etiological factor in patients with burning mouth syndrome.

War-related transformation and work of surgery service of the Pozega Medical Center, East-Croatian Hospital unaffected by direct war activities.

The work of the Pozega Medical Center Surgical Unit during the two war years (from July 16, 1991, to October 1, 1993) in Croatia is described. Since the war operations did not affect the town directly, the work was mostly characterized by treatment of wounds inflicted by explosive devices (51.2%) and localized mostly on the extremities (62.9%). Apart from these wounds, there were 0.7% burns, 26.6% gunshot wounds, and 19.6% injuries caused by blunt and hard objects. Of the total of 959 treated wounded persons, only 18 (1.9%) died, illustrating the success of the therapy administered according to the war surgery doctrine criteria.

Deletion screening of the Duchenne/Becker muscular dystrophy gene in Croatian population.

The dystrophin gene deletion in 53 Duchenne and 21 Becker muscular dystrophy (DMD/BMD) male patients was analyzed by DNA test using multiplex polymerize chain reaction (M-PCR) in Croatian population. The overall percentage of deletion cases observed was 50%; 61% (53/32) for DMD and 38% (21/8) for BMD. The number of deleted exons was variable, but generally DMD deletions involving single-exon 19, 44, 50, 51 and larger exon deletions 3-6, 4-12, 4-17, 8-13, 12-13, 12-19, 48-50, 50-51, 50-52, 51-52 were more frequent. Eight patients with BMD had deletions exon 45-47, 45-48, and exon 3. The results obtained in the present study showed location of breakpoints in the dystrophin gene, and pointed to variability of deletion patterns in Croatian population among different European populations.

Apolipoprotein E genotypes and metabolic risk factors for coronary heart disease in middle-aged women.

Apo E genotypes and plasma metabolic risk factors (total cholesterol, triglycerides, HDL and LDL cholesterol, total/HDL cholesterol ratio, lipoprotein Lp (a), apolipoprotein A-I, A-II, apo B, and apo E) were determined in 134 healthy middle-aged (X +/- SD 49.62 +/- 4.83) women. The aim of this study was to investigate metabolic risk markers according to various apo E genotypes, and to evaluate a possible risk for coronary heart disease. The results revealed that the frequencies of apo E3/3 are the most frequent (46%), followed by E4/4 (2%), E3/4 (14%), E2/3 (14%), and E2/4 (2%) in the middle-aged women. Higher mean triglycerides, LDL-C and apo B levels were found with apo E3/4, and lower mean levels of HDL-C i.e. apo A-I than in other analyzed genotypes. Greater mean of total/HDL ratio and lower levels of apo A-II were seen with E2/4. Serum lipoprotein Lp (a) concentration was higher in women with genotypes E3/3. Apo E concentration was the lowest with genotypes E4/4, i.e. the highest with E2/3. Serum total cholesterol tended to be higher in women with genotypes E4/4. Genotype E3/4 is connected with the highest concentrations of (X +/- SD) triglycerides (1.74 +/- 0.78), LDL (4.28 +/- 1.88), apo B (1.03 +/- 0.32) and with the lowest concentrations of HDL cholesterol (1.11 +/- 0.21) in the relation to the other analyzed genotypes. This group of women could possibly represent high risk women for CHD. Genotype E3/3 is associated with the highest concentration of independent genetic risk marker for CHD, lipoprotein Lp (a) (0.19 +/- 0.27). The genotype E4/4 has the highest concentration of total cholesterol (5.93 +/- 1.01), and has to be taken in account for risk evaluation in women. High level of apo E (0.11 +/- 0.05) and low level of apo A-I (1.80 +/- 0.44) were associated with E2/3 genotypes. The significance of E3/4 with the high total/HDL ratio (5.52 +/- 2.21) and low apo A-II (0.53 +/- 0.09) is important indicator, because total/HDL cholesterol ratio represents independent Established Risk Factor (ERF) for CHD. Apolipoprotein E genotypes as genetic markers and investigation of serum metabolic risk markers appear to be important in view for further evaluation of high risk women for CHD in our population.

Inflammatory mediators in saliva of patients with rapidly progressive periodontitis during war stress induced incidence increase.

Rapidly progressive periodontitis (RPP) results from the interaction between the periodontal microflora and the host. Stress is believed to play an important role in determining host responses, and it has been proposed that hyperactivity of host defense mechanisms significantly increases tissue destruction typical for this disease. During a period of four months we have diagnosed 20 patients with acute RPP, all of them active participants in battles of the Croatian liberation war with posttraumatic stress disorder (PTSD) related symptoms. In these patients we analyzed biochemical parameters in unstimulated saliva and performed microbiological analyses of periodontal pockets. These findings were compared with those of patients with adult periodontitis (AP), edentulous and healthy persons, none of whom participated in the war. Persons with AP had reduced concentrations of host humoral defense factors in saliva (C-reactive protein, C3 component of complement, and aplha alpha 2-macroglobulin), while patients with RPP had increased concentration of interleukin-6 (IL-6). IL-6 is released by host inflammatory cells and is a mediator of bone resorption. Actinobacillus actinomycetemcommitans and Peptostreptococcus were more frequently isolated from patients with RPP. We interpret these results as indicators of the importance of stress in the causation of RPP, with host inflammatory hyperactivity playing an important role in tissue destruction, specially alveolar bone resorption possibly caused by increased local levels of IL-6.

The usefulness of biochemical bone markers in predicting the response of hormone replacement therapy in perimenopausal cigarette smoking healthy women.

The purpose of our study was to evaluate the effects of orally administered combined sequential estradiol (2 mg 17 beta estradiol) with progestin (1 mg norethisteron acetate) daily during ( +/- SD) 15.34 +/- 13.89 months on bone markers in perimenopausal cigarette smoking women. The control group consisted of cigarette smoking perimenopausal women without hormone replacement therapy (HRT). The following biochemical bone markers were analyzed in hormone replacement users (N = 35) and non-users (N = 28): serum total calcium (Ca), total alkaline phosphatase (ALP), procollagen I C-terminal propeptide (PICP), cros-linked carboxyterminal collagen I telopeptide (ICTP) and osteocalcin (OC). When we compared the results of bone markers in the cigarette smoking current users and non cigarette smoking non-users, we found statistically significant lower levels of bone formation markers, ALP and OC, and lower level of bone resorption marker; ICTP in users than in non-users. In perimenopausal cigarette smoking women on HRT lower levels of new biological markers reflected less intensive bone remodelling and probable decrease in bone loss than in non-users. These results indicate that the measurement of biological bone markers are useful to identify risk women for osteoporosis who may have special benefit from the treatment with hormone replacement therapy, even when they smoke.

Changes in vitamin E concentration after surgery and anesthesia.

UNLABELLED: The aim of this randomized study was to examine changes in vitamin E concentration in female subjects (age 30-60, ASA I) after cholecystectomy and halothane (N = 16) or isoflurane (N = 16) anaesthesia. Vitamin E concentration was measured two days before, and then one, five and twenty-four hours and four days after surgery. High-pressure liquid chromatography was used for its determination. Simultaneously activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyltransferase (GGT) were determined. STATISTICAL ANALYSIS: ANOVA, Tukay HSD test. The research has been accepted by the Drugs Committee of the Karlovac County Hospital. Preoperative vitamin E concentrations in the halothane group were 8.69 +/- 2.35 micrograms/L, median 8.67 micrograms/L and in the isoflurane group 9.43 +/- 2.4 micrograms/L, median 9.08 micrograms/L. Statistically lower vitamin E concentrations compared with preoperative values were noted one hour (P < 0.05), 5 hours (P < 0.01), 24 hours (P < 0.01), as well as 4 days (P < 0.01) after the operation. The lowest vitamin E concentrations were noted 24 hours after the operation with statistically insignificantly higher values in the isoflurane group (halothane group 5.98 +/- 2.08 micrograms/L, isoflurane group 6.58 +/- 1.51 micrograms/L). Analyzing enzyme (ALT, AST and GGT) pre- and postoperative values, no statistically significant differences between the investigated groups and during the time were observed. Statistically significant differences were found between individual measurement times, with no statistical significance of the differences between the halothane and isoflurane groups. It seems that neither the difference in halothane and isoflurane biotransformation nor their distinct effect on perfusion of some organs are the determining factors in post-operative changes in vitamin E concentration.

Deletions in the SMN and NAIP genes in patients with spinal muscular atrophy in Croatia.

Two genes, i.e. survival motor neuron (SMN) and neuronal apoptosis inhibitory protein (NAIP) have been mapped to the SMA region of chromosome 5q13. Both genes are frequently deleted or truncated in SMA patients. We have studied 26 patients with SMA types I-III, 29 first relatives, and 14 subjects with mild adult-onset type IV. DNA deletion genotypes were determined by PCR techniques amplifying exons 7 and 8 of SMN, and exon 5 of NAIP gene which distinguish SMN and NAIP telomeric copy from a non-pathogenic gene homologue as a centromeric copy. Results revealed the homozygous deletions of exon 7 and 8 of the SMN gene and exon 5 of the NAIP gene in 3/3 infants with SMA I and in 1/20 with SMA type II. Exons 7 and 8 of the SMN gene were homozygously deleted in 10/20 and only exon 7 in 6/20 children with SMA type II. The overall percentage of deletion cases observed was 77% in children with SMA types I-III. Adult patients with type IV SMA showed no homozygous deletion of exons 7, 8 and 5 of the SMN and NAIP genes. Also, all relatives had both a telomeric and centromeric SMN and NAIP copy. Deletion analysis of SMN and NAIP genes are a significant diagnostic tool, because there are clinical entities resembling SMA which most likely have another pathogenetic background.

War injuries in civilians treated in Pozega Hospital, Croatia.

The management of 131 civilians with war injuries, treated at the Department of Surgery, Pozega County Hospital, during the 1991-1995 period is presented. The majority of 55 (42%) of patients were injured at the front line. There were 75 (57.3%) of patients with explosive injuries 67 (51.2%) involved the extremities, while 29 (22.1%) involved the head. Seven (5.3%) patients died. Amputation of part of an extremity was required in five (3.8%) patients.

Changes in skeletal metabolism in pubertal girls can be revealed by biochemical parameters of calcium metabolism and bone turnover.

Biochemical changes related to skeletal turnover in puberty were investigated in a sample of 67 girls aged 8-14 years. The following biochemical parameters were measured in serum: total calcium, phosphate, magnesium, total alkaline phosphatase, osteocalcin, and calcium and hydroxyproline in the second morning urine. Thirty-five premenarchal girls (8-11 years) had significantly lower serum calcium, and higher alkaline phosphatase and phosphate than those menstruating regularly (N = 32, 12-14 years). A statistically significant negative correlation of serum parameters and age was found for phosphate and alkaline phosphatase in all subjects, and for calcium and magnesium only in the premenarchal girls. These results indicated the more intensive processes of skeletal metabolism occurring in prepubertal age and early puberty to reflect in basic biochemical parameters of calcium and bone metabolism. Analysis of correlation between biochemical parameters showed alkaline phosphatase and phosphate to correlate positively with hydroxyproline excretion and negatively with urinary calcium in all subjects. In the subjects after menarche, osteocalcin correlated with alkaline phosphatase and phosphate. Thus, biochemical parameters indirectly reflected physiologic changes occurring with bone turnover in puberty. Variations in bone turnover during puberty, including a more pronounced bone formation during prepubertal or early stages, can be indirectly observed through biochemical parameters related to calcium and bone metabolism. Investigations of skeletal growth and puberty would benefit from specific markers of bone remodeling and "basic" biochemical parameters, as it might disclose subtle metabolic relationships.

Osteocalcin reference range in a Croatian population sample.

Osteocalcin concentrations were measured in 40 healthy males and 60 healthy females aged 21-90 years by a commercial RIA kit. No significant difference between sexes was found for osteocalcin, and a negative and exponential regression with age existed only for females. In comparison to reference values of other investigators, higher osteocalcin concentrations were observed and considered consequential to sample selection and wide age range. Validity of the established reference range (1-18 ng/mL) was confirmed by measuring osteocalcin in patients with different bone metabolism disorders.

Plasma lipids, lipoprotein Lp(a), apolipoproteins, and hemostatic risk factors distributions in postmenopausal women.

In the present study, the effect of hormone replacement therapy on lipid metabolism, apolipoproteins and hemostatic risk factors for cardiovascular disease was assessed in 216 Croatian postmenopausal women. There were 156 current users divided in to two groups according to the duration of therapy. The short-term study of < 10 months (X +/- SD 5.31 +/- 2.69) included 49 users, and long-term study of > 11 months (X +/- SD 22.06 +/- 10.95) included 107 users of hormone replacement therapy. Sixty nonusers served as a control group. In the short-term study, current users had a significant increase in serum HDL cholesterol, apolipoprotein A-I, A-II and a decrease in total/HDL cholesterol ratio, apoB and antithrombin III (p < 0.05). No significant differences were recorded for total cholesterol, triglycerides, LDL cholesterol, lipoprotein Lp(a) and plasminogen. In the long-term study, a significant increase in HDL cholesterol, apo A-I and total/HDL cholesterol ratio, and a decrease in AT III were observed. Results of the study showed favorable effects of hormone replacement therapy on serum lipid profile and apolipoproteins as a protective regimen from cardiovascular disease in both treatment groups of postmenopausal women. There are conflicting reports regarding increased fibrinolytic activity. The clinical relevance of the observed changes in antithrombin III concentrations as an important coagulation inhibitor is doubtful and should be considered in a more extensive evaluation of the potential hemostatic risk factors for cardiovascular risk and thromboembolism.

Two new mutations (1811 + 1G-->C and Y569C) identified in the CFTR gene in patients of Macedonian and Croatian origin.

Two novel CFTR gene mutations were identified in one patient of the Macedonian and Croatian origin, respectively. The two mutations were detected by the method of denaturing gradient gel electrophoresis (a splicing mutation 1811 + 1G-->C) in intron 11, and by single strand conformation polymorphism analysis (a missence mutation Y569C) in exon 12. The mutations were characterized by direct sequencing of amplified DNA, according to Sanger. These two novel mutations were detected as associated with the delta F508 mutation.

[60 years of organized surgery in Pozega]. / 60 godina organizirane kirurgije u Pozegi.

The 60 years of organized surgery in Pozega started by Dr. Artur Horvat in the old Vucjak hospital is described. This period was designated by work, personnel and two wars occurring 50 years apart. Events and personalities dictated that time to be divided into 5 phases. The significance of that period is detailed by the use of surgical protocols, rare literature and verbal communications.

[Histology of bone tissue and histomorphology--report on standardized terminology]. / Histologija kostanog tkiva i histomorfometrija--prikaz ujednacene terminologije.

The aim of this paper was to present and describe standardized terminology used in histologic and histomorphometric analysis of bone tissue. Bone tissue is characterized by specific activities which are the result of cell function throughout life. Histologic analysis of bone tissue specimen provides an insight in the features and quality of cellular activities. Histomorphometry is direct measurement and calculation of many parameters which permits quantification of characteristics and dynamics of particular bone tissue function. This method may be applied in diagnostics and monitoring of therapy effects in different metabolic bone disorders.

[In situ PCR in the diagnosis of acute leukemia]. / PCR in situ u dijagnostici akutnih leukemija.

Cytomorphologic and cytochemical bone marrow analysis is essential in the diagnosis of acute leukemia. Immunophenotyping and conventional cytogenetics, just as fluorescent in situ hybridization (FISH) are other diagnostic procedures, as well as genome analysis by PCR (polymerase chain reaction). PCR is inevitable in searching for minimal residual disease, because it may detect very small amount of malignant hematopoietic cells even when a patient is in complete remission (less than 5% malignant cells in bone marrow and disappearance from peripheral blood) which helps better monitoring of patients. By in situ hybridization (ISH) it is possible to associate specific cell type with genome alteration, but the method is not sensitive enough. By combining ISH and PCR a novel technique with increased sensitivity was developed, PCR in situ, which enables nucleic acid amplification in an intact cell. In this case report we present two patients whose bone marrow aspirates were analyzed also by PCR in situ.