Steroid hormones in systemic sclerosis: associations with disease characteristics and modifications during scleroderma renal crisis.

OBJECTIVE: The renin-angiotensin-aldosterone system (RAAS) and glucocorticoids (GCs) are involved in vascular remodeling and fibrosis, but have not been extensively studied in systemic sclerosis (SSc). Our aim was to investigate the RAAS and GC hormones in SSc patients. METHODS: Serum levels of renin (dosage and activity), aldosterone and its precursors (DOC, B, 18-OH-DOC, 18-OH-B), and GCs (cortisol, cortisone, 11-deoxycortisol, 18-OH-F) were assessed in 122 SSc patients and 52 healthy controls. After applying stringent inclusion criteria aimed at ensuring accurate hormone assessments (exclusion of interfering drugs, strict sampling conditions), we analyzed RAAS hormones in 61 patients, and GCs in 96 patients. Hormone levels were compared between patients and controls; and associations with disease characteristics were assessed in patients. RESULTS: Regarding RAAS hormones, SSc patients displayed significantly lower aldosterone levels (although within normal range), similar renin levels, and higher B levels than controls. Abnormal RAAS hormone levels were associated with a more severe SSc phenotype (lung and skin fibrosis, heart and pulmonary vascular involvements, inflammation). Regarding GC hormones, SSc patients had higher levels of cortisol, 11-desoxycortisol (precursor) and 18-OH-F (metabolite) but lower levels of cortisone (inactive counterpart) than controls.RAAS hormone levels were assessed in 5 SSc patients before and during scleroderma renal crisis (SRC): concentrations varied considerably between patients, but consistently included normal/increased aldosterone levels and elevated renin levels. CONCLUSION: RAAS and GC hormones are abnormally produced in SSc patients, especially in patients with severe SSc and during SRC. This could suggest a participation of these hormonal systems in SSc pathogenesis.

Accuracy of leptin serum level in diagnosing ventilator-associated pneumonia: a case-control study.

BACKGROUND: Undernutrition causes a reduction of body-fat mass and a decrease in the circulating concentration of leptin which impairs the production of proinflammatory cytokines and increases the incidence of infectious diseases. The main objective of this study was to determine whether leptin deficiency is a risk factor for ventilator-associated pneumonia (VAP). METHODS: This prospective observational case-control study was conducted in a university ICU during a 2-year period. Patients with VAP (cases) were matched (1:1) to patients without VAP (controls) according to all the following criteria: age, gender, SAPS II, and duration of ICU stay before VAP occurrence. In all patients leptin, C-reactive protein (CRP) and procalcitonin (PCT) were measured at ICU admission, and twice a week. In addition, in cases, leptin, CRP and PCT were also measured on the day of VAP diagnosis. RESULTS: Eighty-six cases were matched with 86 controls. No significant difference was found in leptin and PCT levels between cases and controls. CRP level was significantly higher on the day of VAP in cases compared with controls (99 vs. 48 mg/L, P=0.001). Combination of CRP-leptin (CRP ≥78 mg/L and leptin ≥6.2 ng/mL on the day of VAP) was significantly (P=0.009) associated with VAP in univariate analysis. Multivariate analysis identified the combination of CRP-leptin (OR [95% CI] 3.08 [1.18-8.04], P=0.003), LOD score (1.27 [1.08-1.48], P=0.003), neuromuscular-blockers use (6.6 [2.03-21.7], P=0.002), and reintubation (3.3 [1.14-9.6], P=0.027) as independent risk factors for VAP. CONCLUSION: In our study, leptin level was not associated with VAP occurrence. Further studies are needed to confirm our results, and to define the exact inflammatory role of leptin, and its interest as a biomarker in ICU patients.

Phenotype-genotype correlation and follow-up in adult patients with hypokalaemia of renal origin suggesting Gitelman syndrome.

INTRODUCTION: Gitelman syndrome (GS) is a tubulopathy caused by SLC12A3 gene mutations, which lead to hypokalaemic alkalosis, secondary hyperaldosteronism, hypomagnesaemia and hypocalciuria. AIM: The aim of this study was to assess the prevalence of SLC12A3 gene mutations in adult hypokalaemic patients; to compare the phenotype of homozygous, heterozygous and non-mutated patients; and to determine the efficiency of treatment. METHODS: Clinical, biological and genetic data were recorded in 26 patients. RESULTS: Screening for the SLC12A3 gene detected two mutations in 15 patients (six homozygous and nine compound heterozygous), one mutation in six patients and no mutation in five patients. There was no statistical difference in clinical symptoms at diagnosis between the three groups. Systolic blood pressure tended to be lower in patients with two mutations (P=0.16). Hypertension was unexpectedly detected in four patients. Five patients with two mutated alleles and two with heterozygosity had severe manifestations of GS. Significant differences were observed between the three groups in blood potassium, chloride, magnesium, supine aldosterone, 24 h urine chloride and magnesium levels and in modification of the diet in renal disease. Mean blood potassium levels increased from 2.8 ± 0.3, 3.5 ± 0.5 and 3.2 ± 0.3 before treatment to 3.2 ± 0.5, 3.7 ± 0.6 and 3.7 ± 0.3 mmol/l with treatment in groups with two (P=0.003), one and no mutated alleles respectively. CONCLUSION: In adult patients referred for renal hypokalaemia, we confirmed the presence of mutations of the SLC12A3 gene in 80% of cases. GS was more severe in patients with two mutated alleles than in those with one or no mutated alleles. High blood pressure should not rule out the diagnosis, especially in older patients.

[Status of iodine nutrition of children until 1 year: consequences on the thyroid function]. / Statut en iode chez des enfants de moins de 1 an: conséquences sur la fonction thyroïdienne.

INTRODUCTION: Iodine deficiency remains a major public health problem mainly in least-developed countries but also in many industrialized countries. OBJECTIVES: The present study aimed at: (1) evaluating the status of iodine nutrition of children until 1 year in the North region of France; (2) studying risk factors for iodine deficiency; (3) evaluating relationship between iodine deficiency and thyroid disorders. PATIENTS AND METHODS: This prospective study was conducted between 1st january and 31st May 2005 in the children's hospital of Lille (North of France) and all hospitalized children until the age of 1 year were enrolled. Urinary iodine assessment was obtained for 95 (83%) of the 114 infants hospitalized during the study period and TSH value was also determined in 57 (60%) of these 114 infants. RESULTS: Median urinary iodine concentration was 328 microg/L (range: 12-1580). Twenty-four (25%) of 95 infants had a high urinary iodine excretion (urinary iodine greater than 400 microg/L). Nineteen (20%) of the 95 infants were iodine deficient (urinary iodine less than 100 microg/L): severe iodine deficiency (less than 20 microg/L; n=5; 5%), moderate iodine deficiency (20-49 microg/L; n=6; 6%), mild iodine deficiency (50-99 microg/L; n=8; 8%). No relationship was found between iodine status and the following data: age, sex, familial thyroid disease history, term and type of delivery, nutritional status, type of feeding at inclusion, chronic disease, familial socioeconomic status. TSH value was high (greater than 5 microU/mL) in 7 (12%) of the 57 infants. Only 1 of these 7 infants was iodine deficient. Only 1 of the 19 infants with iodine deficiency had a high TSH value. CONCLUSIONS: Iodine status is not optimal in our population of hospitalized children until the age of 1 year. There is no clear relationship between iodine status and thyroid function.

[Assessment of plasma endothelin level measurement in systemic sclerosis]. / Intérêt du dosage plasmatique de l'endothéline au cours de la sclérodermie systémique.

PURPOSE: According to current knowledge, endothelin (ET)-1 plays an important role in the pathogenesis of systemic sclerosis (SSc). We assessed ET plasma levels in SSc patients according to the clinical presentation and the presence of complications such as pulmonary arterial hypertension (PAH). METHODS: Sixty-three consecutive patients with SSc were included. The control group included 17 healthy patients. ET plasma level was determined for all patients. Pulmonary function test and pulmonary high resolution computed tomography were performed in 44 patients and echocardiography in 51 patients, to screen for PAH, always confirmed by a right heart catheterization. RESULTS: ET plasmatic levels were higher in SSc patients than in healthy group subjects but the difference was not significant (3.72+/-1.13 vs 3.40+/-0.71 pmol/l, p=0.27). ET plasmatic levels were significantly higher in patients with PAH than in patients without PAH (4.28+/-0.65 vs 3.62+/-1.07 pmol/l, p=0.04) and in patients with anticentromere antibodies (3.96+/-1.11 vs 3.19+/-1.12 pmol/l, p=0.03). There was a positive linear correlation between ET plasmatic levels and systolic pulmonary arterial pressure (r=0.34, p=0.013). The best cut-off value for ET plasmatic level to discriminate patients affected by PAH was determined by ROC curve method: 4.1 pmol/l (sensibility 85.7%, specificity 66%). CONCLUSION: ET plasmatic levels were higher in SSc patients affected by PAH and patients with anticentromere antibodies. There was a positive linear correlation between ET plasmatic levels and systolic pulmonary arterial pressure. Assessment of ET plasmatic levels for detection and monitoring of pulmonary hypertension during SSc is warranted in larger prospective studies.

Sex hormone-binding globulin, estradiol, and bone turnover markers in male osteoporosis.

The role of estrogen deficiency in male osteoporosis is still under discussion. One hundred five subjects, 65 of them suffering from osteoporosis (mean age, 53.9 years) and 40 age-matched controls were studied. Osteoporosis was defined by a T score < -2.5 in the lumbar spine or at the femoral neck. Forty-one (63.1%) of the subjects had a history of low-energy fractures, involving vertebrae in 33 cases (50.8%). Osteoporosis was considered to be idiopathic in 33 subjects (50.8%) for whom no etiology could be found. We measured levels of total estradiol (pg/ml, with a detection threshold of 4 pg/ml), total testosterone (ng/ml), and their carrier protein, that is, sex hormone-binding globulin (SHBG, pmol/ml). Various markers of bone remodeling were also measured. Two of them provide an estimate of bone formation-osteocalcin (OC) and bone alkaline phosphatase (BAP). Two others evaluate bone resorption-procollagen type I C-terminal telopeptide (ICTP) and serum C-telopeptide of type I collagen (sCTX). There was no significant difference in estradiol levels between controls and osteroporosis patients. We did not find any significant correlation between estradiol levels and spinal bone mineral density (BMD) (r = 0.15, P > 0.05), and the relationship between estradiol levels and BMD at the femoral neck was weak (r = 0.25, P < 0.05). On the other hand, SHBG was significantly higher in the osteoporotic patients than in controls (P < 0.01). This difference persisted after adjustment for body mass index (BMI) and after exclusion of patients with a condition known to increase SHBG levels. Moreover, this carrier protein was negatively correlated with BMD at the femoral neck (r = -0.37, P < 0.01) and at the lumbar spine (r = -0.27, P < 0.05). SHBG also correlates strongly with sCTX (r = 0.37, P < 0.01). Finally, logistic regression analysis showed that serum SHBG concentration was significantly associated with the presence of fractures; the odds ratio of having a fracture was 2.04 [95% confidence interval (CI) 1.2-3.4, P < 0.01] for each increase of 1 standard deviation (SD) in the patient's SHBG level. The stronger relationship was nearly the same for the whole group and for patients with idiopathic osteoporosis. This study therefore suggests that SHBG may play a key role in male patients with idiopathic or secondary osteoporosis. It shows that serum SHBG concentration is increased in middle-aged men with osteoporosis and is correlated with hip, spine BMD, and sCTX levels. Finally, our findings are in agreement with previous studies which suggest that serum SHBG is a new biological marker of fracture risk in men.

Eligibility of diabetic patients receiving dialysis for islet after kidney transplantation.

INTRODUCTION: The aim of this study was to assess the selection of candidates among 38 dialyzed diabetic patients referred between January 1, 1998 and December 31 2002 for kidney followed by islet transplantation (IAK). The main criteria of eligibility for possible IAK were as follows: (1) plasma C-peptide negative; (2) need for a kidney graft; (3) kidney plus whole pancreas transplantation not desired by the patient; and (4) acceptable results of postkidney graft preislet transplantation evaluation. RESULTS: Seventeen of 38 patients with positive C-p diabetes received a kidney graft alone. Among the 21 C-p-negative diabetic patients, 3 were not eligible for kidney transplantation mainly for psychological reasons and 4 were eligible for kidney plus pancreas transplantation. The remaining 14 C-p-negative patients underwent kidney transplantation or had previously undergone kidney transplantation. Among them, 1 had moved away, 1 refused IAK, one had slightly positive stimulation tests, 1 was overweight, 1 had breast cancer, and 1 had postkidney graft complications. Among the remaining 8 of 14 C-p-negative, kidney-engrafted patients listed for IAK, 5 have undergone transplantation, 3 with a pre-Edmonton and 2 with the Edmonton protocol. CONCLUSION: In conclusion among this series of 38 diabetic patients undergoing dialysis, more than 90% were kidney-grafted. Approximately 50% were ineligible for pancreas transplantation or IAK because of a positive C-p, and 20% were enlisted for IAK. These results highlight the importance of C-p determinations in diabetic dialysis patients to identify eligible patients for pancreas transplantation or IAK.

Extreme subcutaneous insulin resistance: a misunderstood syndrome.

Extreme subcutaneous insulin resistance (SIR) is a rare syndrome characterized by severe resistance to subcutaneous insulin with normal intravenous insulin sensitivity. Its pathophysiology is unknown, though an increased insulin degrading activity has been suggested. We report the case of a 35 year-old female patient with type I diabetes since the age of 3. Despite five shots of insulin/day, the patient progressively developed permanent ketosis related to severe acquired SIR with insulin doses as high as 500 U/day. Subcutaneous infusion of insulin and lispro insulin through an external pump did not improve resistance: HbA(1c) levels remained between 14 and 18% (N<6.5%). After numerous ketoacidotic episodes, continuous ambulatory intravenous insulin infusion was attempted through a central port due to a lack of peripheral venous access. HbAlc improved (8.5%) and daily insulin needs decreased to below 40U. However, the treatment had to be discontinued because of thrombosis and infection at different times. Intraperitoneal insulin infusion with an external pump was then proposed. HbAlc improved to 8% during 18 months but several episodes of catheter infection and encapsulation led to its removal. An intraperitoneal pump was surgically implanted, leading to the stabilization of HbA(1c) to around 8%. An insulin degradation assay did not demonstrate any excess of insulin degrading activity in the patient's or controls' subcutaneous tissue; nevertheless, excessive amounts of insulin were found in the patient's derm compared to controls. This case report of acquired SIR raises the question of its treatment and mechanisms. Regarding treatment, intraperitoneal delivery of insulin appears to be the best solution, but the mechanisms underlying SIR still remain unclear.

[Iodine nutritional status and risk factors for iodine deficiency in infants and children of the french North department]. / Statut en iode et facteurs de risque de déficit en iode chez des enfants vus en consultation de protection maternelle et infantile dans le département du Nord.

INTRODUCTION: Iodine deficiency is responsible for a higher mortality and morbidity in neonates and infants. It has not yet disappeared in European countries, especially in Southern and Eastern Europe. OBJECTIVES: The present study aimed at evaluating the status of iodine nutrition of infants living in the North department (France) and at studying risk factors for iodine deficiency. METHODS: The study was conducted in primary health care centres in 160 healthy infants aged ten days to six years (mean +/- SD: 17.7 +/- 2.5 months). Data included: familial thyroid disease history, type of feeding at inclusion, timing of introduction of complementary foods, nutritional status (weight, height, head and arm circumference), as well as maternal education level and family socio-economical status. Iodine status was assessed by urinary iodine excretion. RESULTS: Urinary iodine concentration ranged from 4 to 1042 microg/l (median +/- SD: 195,5 +/- 21,6 microg/l). Thirty-eight (24%) of 160 children were iodine deficient (urinary iodine < 100 microg/l): mild iodine deficiency (50-99 microg/l: 17%), moderate iodine deficiency (20-49 microg/l: 5%), severe iodine deficiency (<20 microg/l: 2%). No relationship was found between iodine status and age, sex, geographic origin of the children, as well as social and occupational group of the parents. Breast-feeding did not prevent from iodine deficiency. Iodine status did not differ between the cow's milk fed group and the group that was not fed cow's milk. Formula feeding was associated with iodine deficiency (p = 0,02). CONCLUSIONS: Prevalence of severe iodine deficiency was very low in this population. However, iodine status was not optimal.

The human chorionic gonadotropin test is more powerful than the gonadotropin-releasing hormone agonist test to discriminate male isolated hypogonadotropic hypogonadism from constitutional delayed puberty.

OBJECTIVE: The effectiveness of biological investigations aiming at discriminating isolated hypogonadotropic hypogonadism (IHH) from constitutional delayed puberty (CDP) in male patients is still controversial. We revisited the diagnostic power of the basal serum testosterone level, the Triptorelin test and the human chorionic gonadotropin (hCG) test in a cohort of 33 boys with delayed puberty. DESIGN: Boys were aged 14.2 to 26.2 Years at referral. A 5-Year-long clinical follow-up after the initial study allowed confirmation of the diagnosis. At the end of the follow-up period, IHH was found in 13 patients while the other 20 had normal spontaneous pubertal development (CDP). RESULTS: At referral, a basal morning testosterone level >1.7 nmol/l was observed in 55% of patients with CDP exclusively (predictive positive value (PPV)=100%; predictive negative value (PNV)=59%). For CDP, the PPV of the LH peak 3 h after Triptorelin was 100% by setting the upper threshold at 14 IU/l and the PNV was 72%. However, no lower threshold could discriminate IHH from CDP in the remaining patients with an LH peak 3 h after Triptorelin <14 IU/l. In CDP patients, the PPV of the serum testosterone increment after hCG stimulation (deltaT/hCG) was 100% for values >9 nmol/l (PNV=72%). In IHH patients, the PPV of deltaT/hCG was 100% for values <3 nmol/l (PNV=82%). Only 29% of the studied population had a deltaT/hCG between these lower and upper thresholds and therefore could not have been classified initially. CONCLUSIONS: (i) Dynamic testing for the diagnosis of delayed puberty is useful only when the basal testosterone level is lower than 1.7 nmol/l; (ii) in that case, the hCG test has better discriminating power than the Triptorelin test and appears as the best cost-effective investigation. It prevents useless and expensive investigations in about one-half of CDP patients with a basal morning testosterone level lower than 1.7 nmol/l.

[Prospective study of early predictive factors of permanent hypocalcemia after bilateral thyroidectomy]. / Etude prospective des facteurs prédictifs précoces de la survenue d'hypocalcémie définitive après thyroïdectomie bilatérale.

AIM OF THE STUDY: The aim of this prospective cohort study was to identify the early criteria potentially predictive for outcome of permanent hypocalcemia after thyroidectomy. PATIENTS AND METHODS: Serum calcium (Ca) et phosphorus (Ph) were measured daily until discharge in 2035 consecutive patients undergoing bilateral thyroidectomy. In all patients experiencing postoperative hypocalcemia, defined as a Ca < 8.0 mg/dl on two consecutive days, parathyroid hormone was measured prior initiation of calcium therapy et discharge (early PTH), et blood sample was also obtained 7 to 14 days after discharge for Ca et Ph measurements (delayed Ca et Ph). These patients were then followed up until complete resolution of hypocalcemia or at least one year. Those still needing substitutive therapy to maintain normocalcemia one year after surgery were considered to have permanent hypocalcemia. Correlation of outcome with clinical characteristics, postoperative Ca et Ph levels, early PTH, et delayed Ca et Ph were examined with univariate analysis et multivariate logistic regression. RESULTS: Postoperative hypocalcemia occurred in 153 patients (7.5%) and spontaneously recovered in all but 7 patients (0.3%). Delayed Ca, and delayed Ph were found to be predictive for outcome of hypocalcemia by univariate analysis (p < 0.01). Relative risk to develop permanent hypocalcemia was 15 for patients with early PTH < 12 pg/ml, 52 when delayed Ph was > 4.0 mg/dl, and 121 when delayed Ca was < 8.0 mg/dl. None of the 113 patients with delayed Ca > or = 8.0 mg/dl and delayed Ph < or = 4.0 mg/dl developed permanent hypocalcemia, in contrast to 1 out of 31 patients (3%) with delayed Ca > 8.0 mg/dl or delayed Ph > 4.0 mg/dl, and 6 out of 9 patients (66%) with delayed Ca < 8.0 mg/dl and delayed Ph > 4.0 mg/dl. Both delayed Ca and delayed Ph appeared as independent factors predicting outcome of hypocalcemia at one year with multivariate logistic regression analysis. CONCLUSION: Delayed serum calcium and phosphorus levels, when measured one week after starting calcium therapy but prior to administration of any vitamin D analogs, accurately predict outcome of hypocalcemia after thyroidectomy. Patients with delayed Ca under 8.0 mg/dl and/or delayed Ph above 4.0 mg/dl are at high risk to develop permanent hypocalcemia.

[Radioimmunoassay of testosterone concentrations in hair: use in doping control]. / Détermination de la testostérone dans les cheveux par une méthode radio-immunologique. Un test pour dépister le dopage?

The measurement of doping agents in hair is attractive since it offers an increase in retrospective detection of doping with these substances. Hair analysis has a wide window of detection, ranging from months to years, depending on the length of the hair shaft and provides information concerning the pattern of an individual's drug abuse. The aim of the present study was to evaluate the ability of a radioimmunological method to determine physiological concentrations of testosterone in hair. Specimens of hair were collected from four children, nine women and twelve men. None of the subjects was an athlete. Hair samples were weighed, decontaminated and digested in sodium hydroxide. The homogenates were extracted by organic solvent. After evaporation, testosterone was measured by radioimmuno-assay. Hair testosterone concentrations ranged 0.6-2.7 pg/mg, 1.8-6.4 pg/mg and 3.6-23.3 pg/mg for children, women and men respectively. This method is fast and accurate and could have applications in doping control, for screening easily numerous hair samples. GCMS is absolutely necessary to confirm results above 30 pg/mg.

Serum levels of inhibins are differentially altered in patients with polycystic ovary syndrome: effects of being overweight and relevance to hyperandrogenism.

OBJECTIVE: To explore the abnormalities of serum inhibin isoform concentrations in a large group of patients with polycystic ovary syndrome (PCOS) and to evaluate the influence of body mass index (BMI), age, LH, and androgens on serum inhibin levels. DESIGN: Prospective study. SETTING: Reproductive endocrinology unit of an academic medical center. PATIENT(S): Forty-one women with PCOS were compared with 24 healthy women. INTERVENTION(S): Blood sampling was performed in the early follicular phase in patients and in control women. MAIN OUTCOME MEASURE(S): Serum levels of inhibin A, inhibin B, alpha-inhibin, pro-alphaC (alpha-inhibin precursor proteins), LH, FSH, E(2), T, and androstenedione (A) were assessed in all subjects. RESULT(S): Serum alpha-inhibin levels together with LH, T, and A levels were significantly increased in women with PCOS. Serum inhibin A levels were lower in patients with PCOS than controls (median +/- SD: 7.35 +/- 2.9 vs. 9.4 +/- 4.7 pg/mL), pro-alphaC levels were higher (264 +/- 136.7 vs. 127 +/- 81.5 pg/mL), and inhibin B levels did not differ between the groups (110.5 +/- 51.5 vs. 108 +/- 47.5 pg/mL). Simple regression analysis showed that inhibin A and B levels were negatively correlated with BMI in patients with PCOS (r = -0.43 and r27 kg/m(2)) displayed significantly lower inhibin A and inhibin B levels and a higher pro-alphaC-inhibin A ratio than nonobese patients with PCOS (BMI

Capillary zone electrophoresis and MALDI-mass spectrometry for the monitoring of in vitro O-glycosylation of a threonine/serine-rich MUC5AC hexadecapeptide.

The in vitro N-acetylgalactosaminylation by human gastric UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases was assessed using the peptide motif GTTPSPVPTTSTTSAP, which is found naturally in the tandem repeat domains of the apomucin encoded by the gene MUC5AC. This peptide appeared to be an excellent tool for obtaining an insight into the extensive O-glycosylation processes of apomucins. Up to six N-acetylgalactosamines were added and the given glycopeptide species were well separated by capillary zone electrophoresis. Moreover, the degree of glycosylation (number of monosaccharide O-linked attachments) could be determined by MALDI-mass spectrometry without prior separation. Using different incubation times, we evidenced the accumulation of various glycopeptides, suggesting that the total glycosylation of an apomucin-peptide requires orderly N-acetylgalactosaminylation processing. This information was completed by experimental data showing that N-acetylgalactosaminylated octapeptides (the peptide backbones of which are part of GTTPSPVPTTSTTSAP) were able to selectively inhibit some N-acetylgalactosaminyltransferases. Our results suggest that this inhibition may influence the quality of the intermediate products appearing during the in vitro O-glycosylation process.

Improved capillary electrophoretic separation of glycosylated oligopeptides through addition of poly(vinyl alcohol), and analysis by electrospray mass spectrometry.

A method for the analysis of O-glycosylation of peptides has been developed, combining capillary electrophoretic (CE) separation and electrospray ionization mass spectrometry. Synthetic peptides with apomucin 'tandem repeat' sequences which present potential O-glycosylation sites on threonine and serine residues were used as model system. In vitro O-glycosylated peptide samples were obtained by incubation of the peptides with human gastric microsomal homogenates containing N-acetylgalactosamine transferase activity in the presence of uridyl diphosphate N-acetylgalactosamine (UDP-GalNAc). CE was carried out in the presence of the linear polymer poly(vinyl alcohol) in the electrophoresis solvent, resulting in a greatly improved separation of the up to five different glycoforms of peptides with lengths of 8, 16 or 23 amino acids, and the unglycosylated peptides. After separation and peak collection, the number of modifications with N-acetyl galactosamine (GalNAc) could be determined by electrospray ionization mass spectrometry. The glycosylation pattern was shown to depend on the amino acid sequence of the peptides.

Influence of the amino acid sequence on the MUC5AC motif peptide O-glycosylation by human gastric UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase(s).

The present work was carried out to study the role of the peptide moiety in the addition of O-linked N-acetylgalactosamineto human apomucin using human crude microsomal homogenates from gastric mucosa (as enzyme source) and a series of peptide acceptors representative of tandem repeat domains deduced from the MUC5AC mucin gene (expressed in the gastric mucosa). Being rich in threonine and serine placed in clusters, these peptides provided several potential sites for O-glycosylation. The glycosylated products were analysed by a combination of electrospray mass spectrometry and capillary electrophoresis in order to isolate the glycopeptides and to determine their sequence by Edman degradation. The O-glycosylation of our MUC5AC motif peptides gave information on the specificity and activity of the gastric microsomal UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase(s). The proline residues and the induced-conformations are of great importance for the recognition of MUC5AC peptides but they are not the only factors for the choice of the O-glycosylation sites. Moreover, for the di-glycosylated peptides, the flanking regions of the proline residues strongly influence the site of the second O-glycosylation.

Polypeptide:N-acetylgalactosaminyltransferase activities towards the mucin MUC5AC peptide motif using microsomal preparations of normal and tumoral digestive mucosa.

The selected-acceptor substrate peptide (TTSAPTTS), deduced from the human mucin gene MUC5AC (expressed essentially in the human gastric and tracheobronchial mucosa), was used to assay polypeptide:N-acetylgalactosaminyltransferases (GalNAc transferases) of different microsomal preparations, obtained from gastric and colonic mucosa in normal and tumoral situations. The O-glycosylated products, analyzed by capillary electrophoresis and electrospray mass spectrometry, showed a variable number of GalNAc O-linked to the different hydroxy amino acids of TTSAPTTS, depending on the tissue studied. Our observations were consistent with the existence of more than one form of GalNAc transferases which were expressed differentially in the gastrointestinal tract (stomach and/or colon). The levels of enzyme activities showed a tissue-specific pattern as they were high in normal colonic tissue and low in colon cancer. On the other hand, in the tumoral gastric tissue (displaying intestinal metaplasia) a high level of GalNAc transferase activities was obtained, similar to that found in the normal colon. Moreover, slight discrepancies (activities and number of O-linked GalNAc) were only detected between normal gastric and tumoral colonic preparations. Thus, the data indicated that the dedifferentiation of the gastric cancer tissue may induce GalNAc transferase activities similar to those in the normal colonic, tissue and that colonic and gastric tissues may contain families of glycosyltransferases involved specifically in reaction towards particular peptide or protein substrates. In addition, the analysis by capillary electrophoresis and electrospray mass spectrometry revealed, in tumoral gastric as well as in normal colonic tissues, a high dipeptidylaminotransferase activity inducing an elongation of TTSAPTTS by dithreonine. This activity was low in normal gastric and tumoral colonic tissues.

Dipeptidyl aminotransferase activity and in vitro O-glycosylation of MUC5AC mucin motif peptides by human gastric microsomal preparations.

The in vitro O-glycosylation reaction of the MUC5AC mucin motif peptide, TTSAPTTS (in one-letter code), was achieved with human gastric microsomal homogenates. The analyses using capillary electrophoresis online coupled with electrospray mass spectrometry and further Edman degradation of the purified products (obtained by capillary electrophoresis at preparative scale) allowed us to distinguish two components at close masses: the addition of a mass of 202 corresponded to an N-terminal elongation of the peptide TTSAPTTS with the dipeptide (TT) and the addition of a mass of 203 corresponded to an N-acetylgalactosamine O-linkage. Using different peptidase inhibitors, a dipeptidyl peptidase/transferase activity was further characterized. A thiol dependence and an inhibition by H-Gly-PheCHN2 (specific to cathepsin C activity) were found. Moreover, besides TTSAPTTS, other MUC5AC motif peptides (GTTPSPVP, TSAPTTS) were also dipeptide donors (GT and TS, respectively) and our results suggested the involvement of a single dipeptidyl peptidase/transferase activity. Finally, this latter activity modified the in vitro GalNAc incorporation rates when using our selected MUC5AC motif peptides. Our study therefore shows that caution must be taken to prevent peptidic substrate elongation while performing in vitro O-glycosylation with microsomal preparations as the enzyme source. In fact, the results of the N-acetylgalactosamine incorporation rates and thus the microsomal N-acetylgalactosamine transferase affinity can be misinterpreted if dipeptidyl peptidase/transferase activity is not inhibited by the thiol inhibitor E-64 or the cathepsin C inhibitor H-Gly-PheCHN2.

O-Glycosylation and cellular differentiation in a subpopulation of mucin-secreting HT-29 cell line.

Malignant transformation of epithelial cells is associated with abnormal glycosylation of mucins. The aim of this work was to evaluate the changes in the O-glycosylation processes during differentiation of tumor cells by performing in vitro reactions using crude microsomal preparations obtained from a subpopulation of HT-29 cells capable of differentiating into mucin-secreting cells (HT-29 MTX cells). The reactions of O-glycosylation were carried out at different times of culture: before confluence (Day 5), when cells are still undifferentiated, and after confluence (Day 21), when cells display a mucin-secreting phenotype. As acceptor for the UDP-N-acetylgalactosamine:polypeptide Nacetylgalactosaminyltransferase (GalNAc transferase), the peptide motif TTSAPTTS (tandem repeat deduced from MUC5AC human gastric gene, expressed in HT-29 MTX cells) was used. A higher rate of enzyme activity was observed in preconfluent cells, and analysis by capillary electrophoresis and electrospray mass spectrometry showed a different pattern of galactosaminylation in pre- and postconfluent cells. Core 1 UDP-galactose:N-acetyl-alpha-galactosaminyl-R 3-beta-galactosyltransferase (3-beta-galactosyltransferase) activityalso decreased with the differentiation, whereas CMP-neuraminic acid:galactose-beta-1, 3-N-acetyl-alpha-galac- tosaminyl-R 3-alpha-sialyltransferase activity increased. In comparison, the evolving process of mucin biosynthesis was tested by the analysis of purified mucins of HT-29 MTX cells, in amino acid and carbohydrate composition, and immunoreactivity assays using several antibodies and lectins. The results suggested that (i) no mucins were detected at Day 5, while the GalNAc transferase and 3-beta-galactosyltransferase activities were already at high rates; (ii) the mucins purified from postconfluent cells showed a high content of sialic acid in an alpha-2,3-linkage to galactose residues; and (iii) cellular differentiation seemed to be accompanied by more regulated processes of glycosylation. This study of the O-glycosylation in HT-29 MTX cells is thus an interesting approach to analyzing the regulation of mucin biosynthesis during cellular differentiation.