Detection of β-ureidopropionase deficiency with HPLC–electrospray tandem mass spectrometry and confirmation of the defect at the enzyme level

The pyrimidine bases uracil and thymine are degraded via the consecutive action of three enzymes to -alanine and -aminoisobutyric acid, respectively. To date, a number of patients have been described with a deficiency of dihydropyrimidine dehydrogenase and dihydropyrimidinase, the first two enzymes of the pyrimidine degradation pathway. In this study, we demonstrate that the first patient presenting with N-carbamyl--amino aciduria, due to a deficiency of -ureidopropionase, was easily diagnosed at the metabolite level using HPLC–tandem mass spectrometry. Urinary analysis showed strongly elevated levels of N-carbamyl--alanine and N-carbamyl--aminoisobutyric acid, with normal or moderately increased levels of the pyrimidine bases and the dihydropyrimidines, respectively. The deficiency of -ureidopropionase was confirmed by measuring all three enzymes of the pyrimidine degradation pathway. No activity of -ureidopropionase could be detected in a liver biopsy of the patient, while a normal activity of dihydropyrimidine dehydrogenase and dihydropyrimidinase was present. Thus, HPLC–tandem mass specrometry proved to be a powerful tool for the initial diagnosis of patients with deficiency of -ureidopropionase.     

Endoprosthesis implantation at the pharyngo-esophageal level： Problems, limitations and challenges

AIM: To present our experience with endoscopic placement of an esophageal endoprosthesis in 19 patients. METHODS: A retrospective evaluation was made for the use of 19 stents positioned at the level of the cervical esophagus: 11 for malignant tumours (7 causing obstruction, 4 complicated by an esophago -tracheal or -cutaneous fistula), and 8 for an acquired benign tracheo-esophageal fistula due to prolonged intubation. The covered Ultraflex stent was used in all cases except two. These two patients had an esophagocutaneous fistula following laryngectomy and a Flamingo Wall stent was used. RESULTS: Stent implantation was technically successful in all patients. Dysphagia score was improved from 3 to 2 in stenosis patients, while sealing of the fistula was achieved in all cases. The median hospital stay was 3 d for malignant tumour patients and 13.5 d for esophagocutaneous fistula patients. One Ultraflex stent and two Flamingo Wall stents were easily removed 33 d and 3 months respectively after implantation when the fistulas had totally occluded. CONCLUSION: Endoprosthesis implantation for malignancy and/or fistula of malignant or benign origin at the level of the cervical esophagus is an easy, well tolerated, safe and effective procedure with no complications or mortality.     

Interaction of glibenclamide and metformin at the level of translation in pancreatic β cells

Sulfonylurea and metformin are used in the treatment of diabetes. Their chronic effects on β cells are not well known. We have shown that sustained exposure of rat β cells to glibenclamide increased their protein synthesis activity, while metformin caused an inhibition. The effect of glibenclamide was attributed to an activation of translation factors. This study examines whether both drugs interact at the level of protein translation in β cells. Purified rat β cells were cultured with and without glibenclamide and metformin before measurement of protein and insulin synthesis, abundance of (phosphorylated) translation factors, and cell viability. A 24?h exposure to metformin stimulated AMP-activated protein kinase (AMPK), suppressed activation of translation factors- both the mammalian target of rapamycin (mTOR; also known as mechanistic target of rapamycin, MTOR)-dependent ones (eukaryotic initiation factor 4E-binding protein 1 and ribosomal protein S6) and the mTOR-independent eukaryotic elongation factor 2-, and inhibited protein synthesis; a 72?h exposure resulted in 50% dead cells. These effects were counteracted by addition of glibenclamide, the action of which was blocked by the mTOR inhibitor rapamycin and the protein kinase A (PKA) inhibitor Rp-8-Br-cAMPs. In conclusion, metformin activates AMPK in β cells leading to suppression of protein translation through mTOR-dependent and -independent signaling. Glibenclamide antagonizes these metformin effects through activation of mTOR- and PKA-dependent signaling pathways.     

Is the increase in serum cystathionine levels in patients with liver cirrhosis a consequence of impaired homocysteine transsulfuration at the level of gamma-cystathionase?

BACKGROUND: It has been suggested that the major metabolic block in the methionine catabolic pathway in cirrhotics exists at the level of the enzyme S-adenosylmethionine synthetase because in previous studies using conventional amino-acid analyzers, no intermediates of transmethylation/transsulfuration were found to accumulate in plasma downstream of S-adenosylmethionine synthesis. We therefore measured serum concentration intermediates of methionine transmethylation/transsulfuration using an improved gas chromatography/mass spectrometry technique. METHODS: Serum concentrations of methionine, homocysteine, cystathionine, N,N-dimethylglycine, N-methylglycine, methylmalonic acid, 2-methylcitric acid and alpha-aminobutyric acid were determined by gas chromatography/mass spectrometry in 108 consecutive patients with liver cirrhosis at Child stages A (mild cirrhosis, n = 27) and B/C (severe cirrhosis, n = 81), 18 outpatients with non-cirrhotic liver disease, and 55 healthy individuals. RESULTS: Serum levels of methionine, N,N-dimethylglycine, N-methylglycine, cystathionine, and homocysteine were significantly higher in patients at Child stages B/C compared with those of healthy controls (P < 0.01), and they were also significantly higher than in patients with non-cirrhotic liver disease (P < 0.01 and P < 0.05 for homocysteine, respectively). They also correlated with the Child-Pugh score (P < 0.01). Homocysteine, cystathionine, N,N-dimethylglycine, N-methylglycine, methylmalonic acid, and 2-methylcitric acid correlated with serum creatinine. The mean cystathionine concentration was significantly higher in patients with creatinine > or = 1.4 mg/dl than in patients with normal creatinine values (P < 0.01). However, the differences between cirrhotics and healthy controls were still significant after correcting for creatinine. CONCLUSIONS: Our data provides indirect evidence for two hitherto unrecognized alterations of methionine metabolism in cirrhotics, i.e. impairment of the transsulfuration of homocysteine at the level of cystathionine degradation and a shift in remethylation of homocysteine towards the betaine-homocysteine-methyltransferase reaction.     

Are we really active in the prevention of obesity and type 2 diabetes at the community level?

The prevalence of type 2 diabetes is increasing rapidly worldwide. Much of this increase in type 2 diabetes epidemic is related to the increase in obesity. There is now firm evidence from randomised trials that type 2 diabetes is preventable by lifestyle modification influencing diet, physical activity and obesity. This prevention effect is sustainable for many years after cessation of active intervention. The slow progression in the development and implementation of population-based strategies in the prevention of obesity and its most common and serious co-morbidity, type 2 diabetes, is of great concern. We summarise published implementation programmes and describe briefly the activities carried out in Finland. In the Finnish implementation programme for the prevention of type 2 diabetes (FIN-D2D), it was found that it is possible to prevent type 2 diabetes "in real life" in the primary health-care settings. We point out that innovative strategic guidelines and their proper implementation are needed to prevent the diabetes epidemic. Among the different tools, also taxation and other regulation to promote healthy food selection and good interaction with the media should be considered.     

Experience in the evaluation of children with hepatosplenomegaly at a teaching ambulatory, São Paulo, Brazil

OBJECTIVES: Describe cases of children with hepatosplenomegaly (HS) attended at the General Pediatric Teaching Ambulatory (AGER) of Instituto da Crian?a, S?o Paulo, identifying the main causes, evolution, necessity for hospitalization and/or referral to specialists. METHODOLOGY: Retrospective analysis of the records of children presenting HS on admission at AGER from September 1, 1993 to August 31, 1996. RESULTS: Of the 89 children included (age range, 1 to 148 months; 24 months), 64 (72%) were referred from other services for HS investigation. Most common presenting complaints were: fever--39 (44%); pallor--26 (29%); weight loss--21 (24%) and jaundice--14 (16%). Main alterations noticed on physical examination were: pallor--47 (53%) and short stature--17 (19%). Anemia was diagnosed in 70 children (79%); 35 children (39%) had infections; 7 (8%) metabolic disorders and 5 (6%) neoplastic disorders. The most frequent infections were of the urinary tract--9 (10%) and hepatitis A--6 (7%). Thirty six children (40%) were referred to specialists, 17 of which were already diagnosed. CONCLUSIONS: Most of the children with HS present deficiency anemia associated with infections which the general pediatrician is able to diagnose. Persistence of unexplained HS for more than 2 months, especially when there is substantial volume enlargement or alteration in the organs consistency, is an indication for referral to specialists.     

Relationship between the functional immunity level and the fluorescent antibody level in mice infected with Plasmodium berghei.

Four groups of mice (Group Ⅰ, 8 mice immunized 3 times before challenge; GroupⅡ, 11 mice, immunized twice; Group Ⅲ, 12 mice, immunized once; Group Ⅳ, 12mice, not immunized) were challenged and then examined with indirect fluorescentantibody test to observe the relationship between the functional immunity level and     

New insights into the biology of multiple myeloma using a combination of May-Grünwald-Giemsa staining and fluorescence in situ hybridization techniques at the single cell level

Up to now only limited information is available about the significance of chromosomal aberrations in multiple myeloma (MM) and about the time point of the neoplastic transformation. Fluorescence in situ hybridization (FISH) combined with standard May-Grünwald-Giemsa (MGG) staining or immunophenotyping on a single cell level were applied. Bone marrow (BM) samples were obtained from 11 patients with morphologically proven multiple myeloma. For detection of the chromosomal aberrations, we used FISH on interphase nuclei with commercially available centromere-specific probes for chromosomes 1, 7, 9, 11, 15, and 17 and further DNA probes for 5p13, 5q31, Rb-gene (13q14), cyclin D1 gene (11q13), and p53 gene (17p13). The aberration rate differed between 14% and 71% on bone marrow smears. Using the combination of MGG and FISH we analyzed eight patients. A total of 2622 bone marrow cells were morphologically identified and investigated for their specific chromosomal aberrations. For all probes applied, 57 cells of the erythropoietic lineage, 698 cells of the granulopoietic lineage, and 168 lymphocytes showed two normal FISH signals. Of 1723 nuclei of plasma cells, 464 (26.9%) were also not aberrant, whereas all other nuclei of plasma cells (n=1259, 73%) showed a specific aberration. Combination of fluorescence immunophenotyping and in situ hybridization (FICTION) was applied in 10 of 11 patients. Seventy-eight investigated CD34-positive precursor cells did not show any specific aberration detected before in the plasma cell compartment. In conclusion, the combination of MGG and FISH on a single cell level demonstrated that only plasma cells bore the chromosomal aberration and MM did not evolve at an early cell level.     

Increased lipoprotein （a） level is independently associated with the severity of coronary artery disease

Background Increased serum level of lipoprotein(a)(Lp(a)) is associated with atherosclerosis. Whether increased Lp(a) level is independently associated with the severity of coronary artery disease(CAD) is unclear. Methods Subjects were enrolled and received coronary angiography to assess the number of stenosed coronary artery. The subjects with CAD were divided into non-significant( 50% stenosis), single and multivessel stenosis(≥ 50 % stenosis) groups. Parameters of interest at baseline were collected. Statistical analyses were performed to evaluate the relationship between Lp(a) level and CAD severity. Results Totally 745 populations were enrolled and diagnosed as CAD(n = 605) or without CAD(n = 140) on the basis of angiography examination. As compared to the subjects without CAD, serum levels of Lp(a) and CRP, and the percentages of subjects with smoking or diabetes were significantly higher in subjects with CAD. In contrast,serum levels of HDL-C and Apo-A were significantly lower in subjects with CAD as compared to subjects without CAD. In comparison of subjects with non-significant stenosis(serum Lp(a) level, 170.0 ± 19.7 mg /d L), serum Lp(a) level was significantly higher in subjects with single(245.5 ± 22.3 mg / d L) or multiple vessel stenoses(265.8 ± 14.0 mg / d L). With multivariate regression analyses, after adjusted for age, gender,smoking, family history and hypertension, there was still significant association between serum Lp(a) level and the number of coronary artery stenosis. After additional adjustment for diabetes, Hb A1 c, total cholesterol,LDL-C, Apo-A, uric acid and CRP, Lp(a) remained strongly associated with CAD severity. Conclusion Serum Lp(a) level was significantly associated with the severity of coronary artery stenosis, which may add the value on cardiovascular risk evaluation.     

Leptin level and its role in the regulation of reproductive function in females with anorexia nervosa

Objective To explore the relationship between serum leptin level and the hypothalamus-pituitary-gonad (HPG) axis in female adolescents with anorexia nervosa (AN). Methods Sixteen newly diagnosed female adolescents with AN in Shanghai and Zhejiang province were investigated, and their serum leptin, FSH, LH and body composition were measured before and after 18 weeks treatment, and their menorrhea cycles were observed. The rhythm of 24 h serum leptin secretion was studied in     

Anaplastic carcinoma associated with a mucinous cystic neoplasm of the pancreas during pregnancy： Report of a case and a review of the literature

Oncogenesis of anaplastic carcinoma of the pancreas is a subject of controversy, because it shows sarcomatous nature with extremely poor prognosis. We herein report an unusual case of anaplastic carcinoma occurring with a recurrent mucinous cystic neoplasm in a 38-year-old female. A 10-cm retroperitoneal cystic mass was pointed out in the first pregnancy and a probable diagnosis of mucinous cystic neoplasm was made in October 2000. She refused surgery first and delivered her baby uneventfully. During her second pregnancy in 2002, however, she presented hematemesis and underwent urgent distal pancreatectomy, splenectomy and partial resection of the gastric wall where the tumor perforated. A diagnosis of borderline-type mucinous cystic neoplasm with ovarian-like stroma was made. Nine months later, CT visualized a recurrent cystic tumor near the pancreatic stump, which was subsequently resected. Pathology revealed that the tumor was composed of two different components of borderline-type mucinous cystic neoplasm and anaplastic carcinoma. The latter was intensely positive for vimentin, CD68, p53 and focally for cytokeratin, suggesting both sarcomatous and carcinomatous differentiation. She survived four years after the second surgery without tumor recurrence. Although the origin of anaplastic carcinoma has not been determined yet, it should be remembered that anaplastic carcinoma can occur in association with mucinous cystic neoplasm of more benign histology.     

Do the Age of Patients with Tetralogy of Fallot at the Time of Surgery and the Applied Surgical Technique Influence the Reoperation Rate?

Abstract Background and Purpose:   Primary repair of tetralogy of Fallot (TOF) has been favored in many centers for years now and results and advantages of this management seem to verify this procedure. The authors wanted to know, if the age at the time of surgery and the surgical techniques had an influence on the long-term results. Patients and Methods:   Between 1992 and 2003, 124 patients underwent complete repair of TOF at the University Hospital Münster, Germany. Patients were subdivided into two groups based on their age (< 1 year and > 1 year of age). Patients in whom a transannular patch (TAP) was used were compared with those without (NTAP), or in whom a conduit was used. Results:   Overall mortality was 8%, with an average age of death of 9.53 years (range 0.06–19.77 years). The patients’ age at the time of surgery affected their survival as only two cases of death were reported among the group of children < 1 year of age (3.2%) whereas eight patients were older (12.9%; p = 0.0483). Six patients died within the first 30 days post surgery. Reoperation had to be performed in 21 cases, 13 (61.9%) of these patients were < 1 year of age at the time of surgery, eight were older (38.1%). A TAP, NTAP or conduit treatment did not show significant differences in long-term survival or freedom from reoperation. Conclusion:   Early repair of TOF within the 1st year of life can be recommended, because mortality is lower than in patients treated at a higher age. There seems no significant difference in the reintervention rate between patients treated within the 1st year of life or later.      

Compatibility of Insulin Lispro,Aspart, and Glulisine with the Solo? MicroPump,a Novel Miniature Insulin Pump

Background

This study compared the stability of commercially available, rapid-acting insulin in the novel tubeless, skin-adhering Solo™ insulin pump over 6 days at extreme environmental conditions.

Methods

Forty-eight pumps for each tested analog were loaded with three different insulin lots and operated at 30 U/day (three sets of 12 pumps) and 15 U/day (one set of 12 pumps) with basal/bolus delivery patterns for 6 days under extreme climatic (37°C, 40% relative humidity) and mechanical (35 strokes/min) stresses. The insulin solutions dispensed were sampled periodically and analyzed for potency, related substances, high molecular weight proteins (HMWP), and preservative content by high-performance liquid chromatography techniques. Biological activity (bioidentity) was demonstrated by an abrupt decrease in blood glucose in rabbits. Solutions were inspected for visual appearance and measured for pH levels.

Results

During the 6-day sampling period, the potency of all insulin samples was maintained at 95.0–105.0% of the bulk solution concentration of the insulin vials. The levels of HMWP and related substances remained well below labeling limits. The preservative concentration decreased with time but remained bacteriostatic effective. Solutions maintained pH and clarity and were particulate free. The biological activity was verified.

Conclusions

Insulin analogs lispro, aspart, and glulisine maintained physical, chemical, and biological properties for 6 days when used in the Solo MicroPump device.