Multiphase computed tomography for localization of parathyroid disease in patients with primary hyperparathyroidism: How many phases do we really need?

BACKGROUND: Multiphase computed tomography (CT) involves multiple cervical CT acquisitions to accurately identify hyperfunctional parathyroid glands, thus increasing radiation exposure to the patient. We hypothesized that only 2 cervical acquisitions, instead of the conventional 4, would provide equivalent localization information and halve the radiation exposure. METHODS: We identified 53 consecutive patients with primary hyperparathyroidism who underwent multiphase CT before parathyroidectomy. All scans were reinterpreted first using 2 phases then using all 4 phases. The accuracies of interpretations were determined with surgical findings serving as the standard of reference. RESULTS: Sixty-four hyperfunctional parathyroid glands were resected with a mean weight of 394.3 mg. Two-phase CT lateralized the hyperfunctional glands in 38 patients with a sensitivity, positive predictive value (PPV), and accuracy of 100%, 71.7%, and 71.7%, respectively. Four-phase CT lateralized the hyperfunctional glands in 39 patients with a sensitivity, PPV, and accuracy of 95.1%, 76.5%, and 73.6%, respectively. For quadrant localization, the accuracy of 2-phase and 4-phase CT was 50.9% and 52.8%, respectively. CONCLUSION: Our results suggest that 2-phase and 4-phase CT provide an equivalent diagnostic accuracy in localizing hyperfunctional parathyroid glands. The reduced radiation exposure to the patient may make 2-phase acquisitions a more acceptable alternative for preoperative localization.

Head and neck cancer.

Cross sectional imaging fills a crucial role in the work up of squamous cell cancer of the head and neck. The radiologist can suggest important considerations in treatment planning and disease prognosis. Key areas of anatomy in radiologic staging are reviewed.

Evaluating 21-day doxycycline and azithromycin treatments for experimental Chlamydophila psittaci infection in cockatiels (Nymphicus hollandicus).

To determine the efficacy of 21-day therapy with azithromycin and doxycycline in the treatment of experimental infection with Chlamydophila psittaci in cockatiels (Nymphicus hollandicus), 30 birds randomly assigned to 3 treatment groups and 1 control group were inoculated with C psittaci by combined intranasal and ocular routes. Morbidity, mortality, and results of polymerase chain reaction testing confirmed that infection was successful. Birds in group 1 (n = 8) received azithromycin at 40 mg/kg PO q48h for 21 days; in group 2 (n = 8), doxycycline at 35 mg/kg PO q24h for 21 days; in group 3 (n = 8), doxycycline at 35 mg/kg PO q24h for 45 days; and, in group 4 (controls; n = 6), no treatment. Six birds died either before or within 2 days of initiating treatment: 4 in the 3 treatment groups and 2 in the control group. Clinical signs resolved and mortality ceased 2-6 days after treatment was initiated in all treatment groups, whereas birds in the control group exhibited clinical signs for the duration of the study. Plasma doxycycline concentrations were measured during the treatment period and exceeded 1 microg/mL at all time points. The absence of clinical signs and mortality in the treatment groups, even after inducing an immunocompromised state with dexamethasone (3 mg/kg IM q24h for 5 days), starting on day 70 postinoculation, suggested that treatment resulted in elimination of the pathogen. After euthanasia of the remaining 24 birds, 23 of the carcasses were submitted for necropsy. Spleen and liver samples from the birds in all treatment and control groups were polymerase chain reaction negative for C psittaci nucleic acid, and organisms were not detected by Gimenez stain. No gross or histologic differences were observed in the livers and spleens of treated and untreated infected birds. Lesions consistent with avian chlamydiosis (hystiocytosis) were seen in all birds and were considered residual. In this study, a 21-day course of either doxycycline or azithromycin was effective in eliminating C psittaci infection in experimentally inoculated cockatiels. Additional studies are necessary to evaluate the efficacy of these treatments in naturally infected cockatiels as well as other species of birds.

Incidental enchondromas of the knee.

OBJECTIVE: The purpose of our study was to determine the prevalence of incidental enchondromas on routine MR knee imaging. MATERIALS AND METHODS: We retrospectively reviewed 449 consecutive routine knee MR examinations for the presence of enchondromas. MRI was considered positive when a focal geographic area of lobular marrow replacement (nonsubchondral) was identified on T1 weighting and high signal intensity was seen on T2 weighting. Patients with enchondromas were further evaluated for demographics; lesion site, size, and relationship to the physeal plate; aggressive imaging features described with chondrosarcoma; concurrent internal derangement; and study indication. RESULTS: The prevalence of incidental enchondromas was 2.9% on routine knee MR examinations. The prevalence was highest in the distal femur (2.0%), followed by the proximal tibia (0.7%) and the proximal fibula (0.2%). The average lesion size was 1.9 x 1.2 x 1.3 cm (57% of lesions were < 1 cm). Most lesions were located in the metaphysis (71%) or diaphysis (21%). Enchondromas were within 1.5 cm of the physeal plate in 72% of cases. No aggressive imaging features to suggest chondrosarcoma were seen. All patients had evidence of internal derangement as the cause of symptoms and the request for imaging. CONCLUSION: Incidental enchondromas can be identified on 2.9% of routine MR knee examinations, most frequently in the distal femur (2.0%). This significant prevalence is much higher than in an autopsy series (0.2%), likely reflecting the increased sensitivity of MRI for detecting small lesions, and is important to recognize to avoid confusion with other pathologic entities.

Tracheal resection and anastomosis in a mallard duck (Anas platyrhynchos) with traumatic segmental tracheal collapse.

A male mallard duck (Anas platyrhynchos) presented for examination for acute respiratory distress and lethargy. The duck had experienced recurrent episodes of respiratory distress since being attacked by a raccoon the previous year, resulting in neck lacerations. Diagnostic tests, including a complete blood count, plasma biochemical analysis, radiography, and tracheoscopy, revealed a collapsed trachea. Surgical correction of the collapsed tracheal segment resulted in resection of 9% of the total tracheal length and subsequent anastomosis. Tracheoscopy performed 2 and 3 months after surgery revealed a healthy mucosa, minimal reduction of the tracheal lumen in the area of anastomosis, and minimal suture granuloma formation.

Biomarker discovery by CE-MS enables sequence analysis via MS/MS with platform-independent separation.

CE-MS is a successful proteomic platform for the definition of biomarkers in different body fluids. Besides the biomarker defining experimental parameters, CE migration time and molecular weight, especially biomarker's sequence identity is an indispensable cornerstone for deeper insights into the pathophysiological pathways of diseases or for made-to-measure therapeutic drug design. Therefore, this report presents a detailed discussion of different peptide sequencing platforms consisting of high performance separation method either coupled on-line or off-line to different MS/MS devices, such as MALDI-TOF-TOF, ESI-IT, ESI-QTOF and Fourier transform ion cyclotron resonance, for sequencing indicative peptides. This comparison demonstrates the unique feature of CE-MS technology to serve as a reliable basis for the assignment of peptide sequence data obtained using different separation MS/MS methods to the biomarker defining parameters, CE migration time and molecular weight. Discovery of potential biomarkers by CE-MS enables sequence analysis via MS/MS with platform-independent sample separation. This is due to the fact that the number of basic and neutral polar amino acids of biomarkers sequences distinctly correlates with their CE-MS migration time/molecular weight coordinates. This uniqueness facilitates the independent entry of different sequencing platforms for peptide sequencing of CE-MS-defined biomarkers from highly complex mixtures.

Predicting the clinical outcome of congenital unilateral ureteropelvic junction obstruction in newborn by urinary proteome analysis.

We analyzed urinary polypeptides from individuals with neonatal ureteropelvic junction (UPJ) obstruction to predict which individuals with this condition will evolve toward obstruction that needs surgical correction. We identified polypeptides that enabled diagnosis of the severity of obstruction and validated these biomarkers in urine collected in a prospective blinded study. Using these noninvasive biomarkers, we were able to predict, several months in advance and with 94% precision, the clinical evolution of neonates with UPJ obstruction.

Discovery and validation of new protein biomarkers for urothelial cancer: a prospective analysis.

BACKGROUND: Non-invasive methods for diagnosis of urothelial carcinoma have reduced specificity in patients with non-malignant genitourinary disease or other disorders. We aimed to use mass spectrometry and bioinformatics to define and validate a cancer-specific proteomic pattern. METHODS: We used capillary-electrophoresis-coupled mass spectrometry to obtain polypeptide patterns from urine samples of 46 patients with urothelial carcinoma and 33 healthy volunteers. From signatures of polypeptide mass, we established a model for predicting the presence of cancer. The model was refined further by use of 366 urine samples obtained from other healthy volunteers and patients with malignant and non-malignant genitourinary disease. We estimated the proportion of correct classifications from the refined model by applying it to a masked group containing 31 patients with urothelial carcinoma, 11 healthy individuals, and 138 patients with non-malignant genitourinary disease. We also sequenced several diagnostic polypeptides for urothelial carcinoma. FINDINGS: We identified a diagnostic urothelial-carcinoma pattern of 22 polypeptide masses. On masked assessment, prediction models based on these polypeptides correctly classified all samples of urothelial carcinoma (sensitivity 100% [95% CI 87-100) and all healthy samples (specificity 100% [84-100]). Correct identification of patients with urothelial carcinoma from those with other malignant and non-malignant genitourinary disease ranged from 86% to 100%. A prominent polypeptide from the diagnostic pattern for urothelial carcinoma was identified as fibrinopeptide A-a known biomarker of ovarian cancer and gastric cancer. INTERPRETATION: Validation of a highly specific biomarker pattern for urothelial carcinoma in a large group of patients with various urological disorders could be used in the diagnosis of other diseases that are identified in urine samples or in other body fluids.

Effects of oral vitamin C supplementation in hemodialysis patients: a proteomic assessment.

Evidence indicates that oxidative stress is present in dialysis patients, and is associated with vitamin C deficiency. Limited data are available regarding the effects of vitamin C supplementation on oxidative stress and inflammation markers in these patients. Moreover, there are no data available on plasma polypeptide fingerprints by proteome analysis before and after vitamin C supplementation. Therefore, we analyzed plasma samples from a prospective, randomized, open-labeled trial to assess the effects of oral vitamin C supplementation (250 mg three times per week), to define the plasma polypeptide pattern in hemodialysis patients. Our results reveal that more than 30 polypeptides show significant changes in the dialysis patients in comparison to controls with normal renal function, and that several polypeptides are affected/normalized by oral vitamin C supplementation. These results underline the remarkable potential for proteomics to recognize specific peptide profiles in different pathological situations, which might not be detected by classical methods.

Combined top-down and bottom-up mass spectrometric approach to characterization of biomarkers for renal disease.

Here we describe a mass spectrometry (MS) approach for biomarker discovery and structural characterization, based on both top-down and bottom-up analyses. Capillary electrophoresis (CE) coupled to electrospray ionization (ESI) time-of-flight (TOF) MS serves to separate and mass-measure the thousands of polypeptides contained in human urine. Statistical analysis of the differences between healthy control samples and patients with focal-segmental glomerulosclerosis, membranous glomerulonephritis, minimal change disease, IgA nephropathy, and diabetic nephropathy validates multiple biomarkers for the control and each of the diseases. To identify those biomarkers, we employ preparative CE, enabling direct infusion ESI MS analysis, followed by sample manipulation and reanalysis where necessary. We show how tandem Fourier transform ion cyclotron resonance (FT-ICR) MS identifies these sometimes large (>8 kDa) biomarkers. Critically, we maintain connectivity between the CE TOF MS data and the ICR data used for biomarker identification.

Detection of acute tubulointerstitial rejection by proteomic analysis of urinary samples in renal transplant recipients.

This study investigates proteomic analysis of urinary samples as a non-invasive method to detect acute rejection of renal allografts. Capillary electrophoresis coupled to mass spectrometry (CE-MS) was used to analyze urinary samples in 19 patients with different grades of subclinical or clinical acute rejection (BANFF Ia to IIb), 10 patients with urinary tract infection and 29 patients without evidence of rejection or infection. A distinct urinary polypeptide pattern identified 16 out of 17 cases of acute tubolointerstitial rejection, but was absent in two cases of vascular rejection. Urinary tract infection resulted in a different polypeptide pattern that allowed to differentiate between infection and acute rejection in all cases. Potentially confounding variables such as acute tubular lesions, tubular atrophy, tubulointerstitial fibrosis, calcineurin inhibitor toxicity, proteinuria, hematuria, allograft function and different immunosuppressive regimens did not interfere with test results. Blinded analysis of samples with and without rejection showed correct diagnosis by CE-MS in the majority of cases. Detection of acute rejection by CE-MS offers a promising non-invasive tool for the surveillance of renal allograft recipients. Further investigation is needed to establish polypeptide patterns in vascular rejection and to explore whether changes in the urinary proteome occur before the onset of histologically discernible rejection.

Pilot study of capillary electrophoresis coupled to mass spectrometry as a tool to define potential prostate cancer biomarkers in urine.

We describe the use of capillary electrophoresis (CE) coupled with mass spectrometry (MS) to identify single polypeptides and patterns of polypeptides specific for prostate cancer (CaP) in human urine. Using improved sample preparation methods that enable enhanced comparability between different samples, we examined samples from 47 patients who underwent prostate biopsy. Of this group, 21 patients had benign pathology and 26 with CaP, and these were used to define potential biomarkers, which allow discrimination between these two states. In addition, CE-MS data from these 47 urine samples were compared to that of 41 young men (control) without known or suspected clinical CaP to further confirm the polypeptides indicative for CaP. Upon crossvalidation of the same samples, several polypeptides were selected that enabled correct classification of the CaP patients with 92% sensitivity and 96% specificity. We then examined an additional 474 samples from patients with renal disease enrolled in other studies and found that 14 (3%) had polypeptides suggestive of CaP possibly indicating that they harbor clinical CaP. In conclusion, this early pilot study suggests that CE-MS of urine warrants further investigation as a tool that can identify putative biomarkers for CaP.

Impact of diabetic nephropathy and angiotensin II receptor blockade on urinary polypeptide patterns.

BACKGROUND: New insights into the pathogenesis and treatment of diabetic renal disease may emerge from recent advances in proteomics using high-throughput mass spectrometry (MS) of urine. METHODS: Using a combination of online capillary electrophoresis (CE) and MS we evaluated urinary polypeptide patterns in four groups of type 2 diabetic patients matched for age, gender, and diabetes duration, including 20 normoalbuminuric patients with and 20 without diabetic retinopathy, 20 microalbuminuric patients with diabetic retinopathy, and 18 macroalbuminuric patients with diabetic retinopathy. Furthermore, changes in urinary polypeptide patterns during treatment with the angiotensin II receptor blocker (ARB) candesartan were evaluated in the macroalbuminuric patients in a randomized double-blinded, cross-over trial where each patient received treatment with placebo, candesartan 8, 16, and 32 mg daily each for 2 months. RESULTS: Overall, 4551 different polypeptides were found in the samples. Urinary polypeptide patterns were comparable in normo- (with and without diabetic retinopathy) and microalbuminuric patients, whereas distinct differences were found in macroalbuminuric patients. Differences in urinary polypeptide patterns between normo- and macroalbuminuric patients permitted the establishment of a "diabetic renal damage" pattern consisting of 113 polypeptides. Eleven of these polypeptides had been sequenced and identified. Candesartan treatment in macroalbuminuric patients significantly changed 15 of the 113 polypeptides in the diabetic renal damage pattern toward levels in normoalbuminuric patients. Change in the diabetic renal damage pattern was not candesartan dose-dependent but individual changes correlated with changes in urinary albumin excretion at each dose level. CONCLUSION: CE-MS serves as a fast and sensitive tool for identification of biomarkers and urinary polypeptide patterns specific for macroalbuminuric type 2 diabetic patients and may be used to explore and monitor renoprotective effects of ARB.

Urine protein patterns can serve as diagnostic tools in patients with IgA nephropathy.

BACKGROUND: IgA nephropathy (IgAN) is the most common chronic glomerular disease in adults. End-stage renal disease (ESRD) develops in about 30% of the patients. Early intervention and consequent therapy may prevent or at least delay the development of ESRD in these patients. Up to now, the diagnosis could only be achieved with a renal biopsy. METHODS: The urine of 45 patients with IgAN was collected and screened for protein/polypeptide patterns with a novel high throughput method, capillary electrophoreses on-line coupled to a mass spectrometer (CE-MS). CE-MS allows the fast and accurate evaluation of up to 2000 polypeptides in one urine sample. The results in IgAN were compared to findings in 13 patients with membranous nephropathy (MN) and 57 healthy individuals. RESULTS: In the patients with IgAN, even when urinary protein excretion was within the normal range of regular tests, the polypeptide pattern in urine differed significantly from that of healthy controls and patients with MN, indicating a specific "IgAN" pattern of polypeptide excretion. Classification regarding discrimination of IgAN from healthy controls and from MN had a sensitivity of 100% and 77%, respectively. Specificity was 90% and 100%, respectively. Compared to patterns established earlier in patients with minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), or diabetic nephropathy (DN), sensitivity and specificity were 100%. Treatment of the patients was associated with changes of the pattern, possibly indicating a therapeutic effect. CONCLUSION: Proteomic analysis with CE-MS coupling permits fast and accurate identification and differentiation of polypeptide patterns in the urine of patients with IgAN, allowing differentiation from healthy controls and, probably, other renal diseases.

Discovery of biomarkers in human urine and cerebrospinal fluid by capillary electrophoresis coupled to mass spectrometry: towards new diagnostic and therapeutic approaches.

We report on our results using capillary electrophoresis coupled to mass spectrometry (CE-MS) to examine human bodyfluids. To demonstrate the versatility of this approach, data on two different bodyfluids, urine and cerebrospinal fluid, are shown. CE-MS analysis of human urine enables the identification of a series of polypeptides which serve as biomarkers for a variety of different renal diseases. The polypeptides are utilized to generate disease-specific polypeptide patterns. Diagnosis of these diseases is possible based on these polypeptide patters. Further, due to the high mass accuracy, polypeptides of interest can subsequently be identified using tandem MS (MS/MS) analysis. The patterns, which are based on distinct polypeptides, allow differentiation of even similar diseases like focal-segmental glomerulosclerosis (FSGS) and minimal change disease (MCD). We present preliminary data suggesting that the indicative polypeptides also enable to evaluate therapy success. Initial data obtained on human cerebrospinal fluid strongly suggest that CE-MS analysis of low-molecular-weight proteins and peptides reveals several potential biomarkers for schizophrenia as well as Alzheimer's disease. In conclusion, the data presented here indicate that CE-MS analysis, applied towards different human bodyfluids, holds the promise to allow diagnosis, staging, and evaluation of therapy success of a large number of diseases, due to its ability to display ca. 1000 individual native polypeptides within ca. 60 min.

A three-step purification strategy for isolation of hamster TIG2 from CHO cells: characterization of two processed endogenous forms.

We have recently isolated a bioactive, circulating protein of human tazarotene-induced gene-2 (TIG2) as the natural ligand of the orphan receptor ChemR23. Here we describe a simplified method for the isolation of hamster TIG2 protein from Chinese hamster ovary (CHO) cell supernatant. Using a heparin-affinity column followed by two reversed phase chromatography steps resulted in the isolation of pure biologically active material. Two processed bioactive forms of Chinese hamster TIG2 were identified by Edman sequencing and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) mass fingerprint analysis, representing the amino acid residues T20 to F156, and T20 to A155 of the 163 amino acid propeptide. Comparison with the predicted aa-sequence indicates a mutation or modification within the C-terminal end of the peptide.

Proteomics: a novel tool to unravel the patho-physiology of uraemia.

BACKGROUND: Uraemic toxicity results in the dysfunction of many organ systems, provoking an increase in morbidity and mortality. To date, only approximately 90 uraemic retention solutes have been described. To examine unknown uraemic substances thoroughly, the identification of as many compounds as possible in the ultrafiltrate and/or plasma of patients would lead to a less biased definition of the uraemic retention process compared with what is proposed today. METHODS: We describe the application of a novel proteomic tool for the identification of a large number of molecules present in ultrafiltrate from uraemic and normal plasma obtained with high- or low-flux membranes. Separation by capillary electrophoresis was coupled on-line to a mass spectrometer, yielding identification of polypeptides based on their molecular weight. RESULTS: Between 500 and >1000 polypeptides with a molecular weight ranging from 800 to 10,000 Da could be detected in individual samples, and were identified via their mass and their particular migration time in capillary electrophoresis. In ultrafiltrate from uraemic plasma, 1394 polypeptides were detected in the high-flux vs 1046 in the low-flux samples, while in ultrafiltrate from normal plasma, 544 polypeptides vs 490 were found in ultrafiltrate from normal plasma obtained from membranes with comparable cut-off. In addition, polypeptides >5 kDa were virtually only detected in the uraemic ultrafiltrate from the high-flux membrane (n = 28 vs n = 5 with the low-flux membrane). To demonstrate the feasibility of further characterizing the detected molecules, polypeptides present exclusively in uraemic ultrafiltrate were chosen for sequencing analyses. A 950.6 Da polypeptide was identified as a fragment of the salivary proline-rich protein. A 1291.8 Da fragment was derived from alpha-fibrinogen. CONCLUSION: The data presented here strongly suggest that the application of proteomic approaches such as capillary electrophoresis and mass spectrometry will result in the identification of many more uraemic solutes than those known at present. This could enable the introduction of more direct elimination strategies, since it is possible to obtain an extended appreciation of the removal capacities of particular dialyser membranes.

Proteomic analysis for the assessment of diabetic renal damage in humans.

Renal disease in patients with Type II diabetes is the leading cause of terminal renal failure and a major healthcare problem. Hence early identification of patients prone to develop this complication is important. Diabetic renal damage should be reflected by a change in urinary polypeptide excretion at a very early stage. To analyse these changes, we used an online combination of CE/MS (capillary electrophoresis coupled with MS), allowing fast and accurate evaluation of up to 2000 polypeptides in urine. Employing this technology, we have examined urine samples from 39 healthy individuals and from 112 patients with Type II diabetes mellitus and different degrees of albumin excretion rate. We established a 'normal' polypeptide pattern in the urine of healthy subjects. In patients with Type II diabetes and normal albumin excretion rate, the polypeptide pattern in urine differed significantly from normal, indicating a specific 'diabetic' pattern of polypeptide excretion. In patients with higher grade albuminuria, we were able to detect a polypeptide pattern indicative of 'diabetic renal damage'. We also found this pattern in 35% of those patients who had low-grade albuminuria and in 4% of patients with normal albumin excretion. Moreover, we could identify several of the indicative polypeptides using MS/MS sequencing. We conclude that proteomic analysis with CE/MS permits fast and accurate identification and differentiation of polypeptide patterns in urine. Longitudinal studies should explore the potential of this powerful diagnostic tool for early detection of diabetic renal damage.

Analytical procedures for quantification of peptides in pharmaceutical research by liquid chromatography-mass spectrometry.

Peptide quantification by liquid chromatography-mass spectrometry (LC-MS) combines the high resolving power of reversed-phase (RP) chromatography with the excellent selectivity and sensitivity of mass spectrometric detection. On the basis of comprehensive practical experience in the analysis of small molecules, pharmaceutical research is developing technologies for analysis of a growing number of peptidic drug candidates. This article is a detailed review of procedures based on LC-MS techniques for quantitative determination of peptides. With the focus on pharmaceutical applications several technologies for sample preparation, various aspects of peptide chromatography, important characteristics of ESI-MS, selectivity of MS-detection modes, the large variability of internal standards, and modern instrumentation are discussed. The demand for reliable, robust, sensitive, and accurate methods is discussed using numerous examples from the literature, complemented by experiments and results from our laboratory.