Effect of vitamin D3 supplementation on vascular and metabolic health of vitamin D-deficient overweight and obese children: a randomized clinical trial.

BACKGROUND: Obese children are vulnerable to vitamin D deficiency and impaired cardiovascular health; vitamin D replenishment might improve their cardiovascular health. OBJECTIVES: The aims were to determine, in vitamin D-deficient overweight and obese children, whether supplementation with vitamin D3 1000 or 2000 IU/d is more effective than 600 IU/d in improving arterial endothelial function, arterial stiffness, central and systemic blood pressure (BP), insulin sensitivity (1/fasting insulin concentration), fasting glucose concentration, and lipid profile and to explore whether downregulation of adipocytokines and markers of systemic inflammation underlies vitamin D effects. METHODS: We conducted a randomized, double-masked, controlled clinical trial in 225 10- to 18-y-old eligible children. Change in endothelial function at 6 mo was the primary outcome. RESULTS: Dose-response increases in serum 25-hydroxyvitamin D concentrations were significant and tolerated without developing hypercalcemia. Changes at 3 and 6 mo in endothelial function, arterial stiffness, systemic-systolic BP, lipids, and inflammatory markers did not differ between children receiving 1000 or 2000 IU vitamin D and children receiving 600 IU. Some secondary outcomes differed between groups. Compared with the 600-IU group, central-systolic, central-diastolic, and systemic-diastolic BP was lower at 6 mo in the 1000-IU group [-2.66 (95% CI: -5.27, -0.046), -3.57 (-5.97, -1.17), and -3.28 (-5.55, -1.00) mm Hg, respectively]; insulin sensitivity increased at 3 and 6 mo and fasting glucose concentration declined at 6 mo (-2.67; 95% CI: -4.88, -0.46 mg/dL) in the 2000-IU group. CONCLUSIONS: Correction of vitamin D deficiency in overweight and obese children by vitamin D3 supplementation with 1000 or 2000 IU/d versus 600 IU/d did not affect measures of arterial endothelial function or stiffness, systemic inflammation, or lipid profile, but resulted in reductions in BP and fasting glucose concentration and in improvements in insulin sensitivity. Optimization of children's vitamin D status may improve their cardiovascular health. This trial was registered at clinicaltrials.gov as NCT01797302.

The final intraoperative parathyroid hormone level: how low should it go?

BACKGROUND: In minimally invasive surgery for primary hyperparathyroidism (HPT), intraoperative parathyroid hormone (IOPTH) monitoring assists in obtaining demonstrably better outcomes, but optimal criteria are controversial. METHODS: The outcomes of 1,108 initial parathyroid operations for sporadic HPT using IOPTH monitoring from 1997 to 2011 were stratified by final post-resection IOPTH level. All patients had adequate follow-up to verify cure. RESULTS: With mean follow-up of 1.8 years (range 0.5-14.3 years), parathyroidectomy using IOPTH monitoring failed in 1.2 % of cases, with an additional 0.5 % incidence of long-term recurrence at a mean of 3.2 years (range 0.8-6.8 years) postoperatively. Operative success was equally likely with a final IOPTH drop to 41-65 pg/mL vs ≤40 pg/mL (p = 1). In the 76 patients with an elevated baseline IOPTH level that did not drop to ≤65 pg/mL, surgical failure was 43 times more likely than with a drop into normal range (13 vs. 0.3 %; p < 0.001). When the final IOPTH level dropped by >50 % but not into the normal range, surgical failure was 19 times more likely (3.8 vs. 0.2 %; p = 0.015). Long-term recurrence was more likely in patients with a final IOPTH level of 41-65 pg/mL than with a level ≤40 pg/mL (1.2 vs. 0; p = 0.016). CONCLUSIONS: Adjunctive intraoperative PTH monitoring facilitates a high cure rate for initial surgery of sporadic primary hyperparathyroidism. A final IOPTH level that is within the normal range and drops by >50 % from baseline is a strong predictor of operative success. Patients with a final IOPTH level between 41-65 pg/mL should be followed beyond 6 months for long-term recurrence.

Development and implementation of an electronic interface for complex clinical laboratory instruments without a vendor-provided data transfer interface.

BACKGROUND: Clinical pathology laboratories increasingly use complex instruments that incorporate chromatographic separation, e.g. liquid chromatography, with mass detection for rapid identification and quantification of biochemicals, biomolecules, or pharmaceuticals. Electronic data management for these instruments through interfaces with laboratory information systems (LIS) is not generally available from the instrument manufacturers or LIS vendors. Unavailability of a data management interface is a limiting factor in the use of these instruments in clinical laboratories where there is a demand for high-throughput assays with turn-around times that meet patient care needs. MATERIALS AND METHODS: Professional society guidelines for design and transfer of data between instruments and LIS were used in the development and implementation of the interface. File transfer protocols and support utilities were written to facilitate transfer of information between the instruments and the LIS. An interface was created for liquid chromatography-tandem mass spectroscopy and inductively coupled plasma-mass spectroscopy instruments to manage data in the Sunquest(®) LIS. RESULTS: Interface validation, implementation and data transfer fidelity as well as training of technologists for use of the interface was performed by the LIS group. The technologists were familiarized with the data verification process as a part of the data management protocol. The total time for the technologists for patient/control sample data entry, assay results data transfer, and results verification was reduced from approximately 20 s per sample to <1 s per sample. Sample identification, results data entry errors, and omissions were eliminated. There was electronic record of the technologist performing the assay runs and data management. CONCLUSIONS: Development of a data management interface for complex, chromatography instruments in clinical laboratories has resulted in rapid, accurate, verifiable information transfers between instruments and LIS. This has eliminated manual data entry that is prone to errors and enabled technologists to focus on analytical applications on the instruments.

Beta-catenin activation promotes liver regeneration after acetaminophen-induced injury.

Acute liver failure (ALF) remains a disease with poor patient outcome. Improved prognosis is associated with spontaneous liver regeneration, which supports the relevance of exploring 'regenerative' therapies. Therefore, the role of the Wnt/beta-catenin pathway in liver regeneration following ALF was investigated. ALF was induced in mice by acetaminophen overdose, which is also a leading cause of liver failure in patients. beta-catenin distribution was also studied in liver sections from acetaminophen-induced ALF patients. A nonlethal dose of acetaminophen, which induces liver regeneration, led to stabilization and activation of beta-catenin for 1 to 12 hours. These data were also verified by increased expression of the beta-catenin surrogate target glutamine synthetase. Beta-catenin activation occurred secondary to the inactivation of glycogen synthase kinase-3beta and an increase in levels of casein kinase 2alpha, and led to increased cyclin-D1, another known beta-catenin target. These observations were next substantiated in beta-catenin conditional-null mice (beta-catenin-null), which show dampened regeneration after acetaminophen injury following induction of CYP2e1/1a2 expression. In light of decreased acetaminophen injury in beta-catenin-null mice despite CYP induction, equitoxic studies in control mice were performed. Significant differences in regeneration persisted following comparable injury in beta-catenin-null and control animals. Retrospective analysis of liver samples from acetaminophen-overdose patients demonstrated a positive correlation between nuclear beta-catenin, proliferation, and spontaneous liver regeneration. Thus, our studies demonstrate early activation of beta-catenin signaling during acetaminophen-induced injury, which contributes to hepatic regeneration.

Toxicology laboratory analysis and human exposure to p-chloroaniline.

INTRODUCTION: p-Chloroaniline is more potent at producing methemoglobin than aniline in animal models. This case highlights the clinical presentation of an inhalation exposure to p-chloroaniline and associated laboratory analysis. An in-vitro study evaluating the metabolism of p-chloroaniline in human hepatocytes was undertaken to evaluate the metabolic fate more closely. CASE PRESENTATION: A 20 year-old man was working at a chemical waste plant when he developed dizziness, abdominal pain, and nausea. The exam was remarkable for coma, tachycardia, cyanosis, and pulse oximetry of 75%. Arterial blood gases showed a pH 7.38, pCO(2) 41 mmHg, pO(2) 497 mmHg, bicarbonate 24 mEq/L and methemoglobin 69%. Methylene blue administration led to complete recovery without sequelae. p-Chloroaniline was later identified as the chemical involved. He denied direct contact with the chemical, but was not wearing a dust mask or respirator. GC/MS confirmed p-chloroaniline and metabolites in the patient's urine. METHODS: Human hepatocytes were incubated with 100 microM p-chloroaniline for 24 hours, in both rifampicin- and vehicle only-treated cells. The cell culture medium was collected for GC/MS analysis for p-chloroaniline metabolites. RESULTS: Similar to the patient sample, both p-chloroaniline and p-chloroacetanilide were identified by GC/MS in hepatocytes incubated with p-chloroaniline. Neither p-chloroaniline incubated in empty cell culture nor direct GC/MS injection of p-chloroaniline generated any p-chloroacetanilide via non-enzymatic degradation. DISCUSSION/CONCLUSION: The seemingly innocuous dermal and inhalation exposure to p-chloroaniline dust can lead to life-threatening methemoglobinemia. The diagnosis can be confirmed with GC/MS analysis of the patient's urine, searching for p-chloroaniline and its primary metabolite p-chloroacetanilide.

Can a lightbulb sestamibi SPECT accurately predict single-gland disease in sporadic primary hyperparathyroidism?

BACKGROUND: Technetium-99m sestamibi scintigraphy with single photon emission computed tomography (SPECT) is widely used to guide minimally invasive exploration in patients with sporadic primary hyperparathyroidism (SPH), although its sensitivity in multiglandular disease is limited. We examined the incidence of missed multiglandular disease and associated anatomic findings when sestamibi SPECT was positive for a single intense focus of delayed tracer uptake, termed a lightbulb scan (LBS). METHODS: Prospectively entered data from 764 patients with SPH treated with initial parathyroid exploration from March 5, 2000, to December 31, 2006, were reviewed. A single radiologist performed blinded interpretation of 585 available sestamibi SPECT images, classifying 167 (28.5%) patients with a LBS. Clinical findings were compared among LBS patients with a single adenoma (true positive) and LBS patients with multiglandular disease (false negative). RESULTS: One hundred fifty of 167 (89.8%) LBS patients had a single adenoma and 3 (1.8%) had carcinoma. Multiglandular disease was anatomically present in 14 of 167 (8.4%) LBS patients compared with 60 of 418 (15.6%) non-LBS patients (p=0.05). Parathyroid hyperplasia occurred less frequently in LBS patients [5/167 (3%)] compared with non-LBS patients [36/418 (8.6%)], (p=0.02), while double adenomas occurred equally often in LBS patients [9/167 (5.4%)] compared with non-LBS patients [24/418 (5.7%)], (p=0.87). Double adenomas in LBS patients were more likely ipsilateral (7/9, p=0.005) and left-sided (7/7, p=0.008). LBS patients with multiglandular disease were more likely to have a history of neck irradiation, prior neck exploration, and concomitant thyroid pathology. CONCLUSIONS: In patients with SPH, sestamibi SPECT studies show a single bright focus of uptake in only 29% of patients. LBS findings do not exclude multiglandular disease. To avoid unacceptable rates of failure at initial parathyroid exploration, the expert surgeon should use validated adjuncts such as intraoperative PTH monitoring or four-gland exploration.

Drug monitoring: simultaneous analysis of lamotrigine, oxcarbazepine, 10-hydroxycarbazepine, and zonisamide by HPLC-UV and a rapid GC method using a nitrogen-phosphorus detector for levetiracetam.

A high-performance liquid chromatography (HPLC) assay using UV detection is described for the simultaneous measurement of the newer generation anti-epileptic medications lamotrigine, oxcarbazepine (parent drug and active metabolite 10- hydroxycarbazepine), and zonisamide. Detection of all four compounds can be done at 230 nm; however, there is a potential interference with zonisamide in patients on clonazepam therapy. Therefore, the method uses dual wavelength detection: 230 nm for oxcarbazepine and 10-hydroxycarbazepine and 270 nm for lamotrigine and zonisamide. In addition, a simple gas chromatography method using a nitrogen-phosphorus detector is described for the measurement of levetiracetam, another of the recently approved anti-epileptic medications. For both methods, limits of quantitation, linearities, accuracies, and imprecisions cover the therapeutic range for drug monitoring of patients. A wide variety of clinical drugs, including other anti-epileptic drugs, do not interfere with these assays. These procedures would be of special interest to clinical laboratories, particularly due to the limited availability of immunoassays for newer generation anti-epileptic medications and that therapeutic uses of these drugs are expanding beyond epilepsy to other neurologic and psychiatric disorders.

Simultaneous determination of lamotrigine, zonisamide, and carbamazepine in human plasma by high-performance liquid chromatography.

A high-performance liquid chromatography assay with ultraviolet detection was developed for the simultaneous determination of the anti-epileptic drugs lamotrigine, carbamazepine and zonisamide in human plasma and serum. Lamotrigine, carbamazepine, zonisamide and the internal standard chloramphenicol were extracted from serum or plasma using liquid-liquid extraction under alkaline conditions into an organic solvent. The method was linear in the range 1-30 microg/mL for lamotrigine, 2-20 microg/mL for carbamazepine, and 1-40 microg/mL for zonisamide. Within- and between-run precision studies demonstrated coefficient of variation <10% at all tested concentrations. Other anti-epileptic medications tested did not interfere with the assay. The method is appropriate for determining lamotrigine, carbamazepine and zonisamide serum or plasma concentrations for therapeutic monitoring.

Serum phosphate and lactate vary with FSH in an early postmenopausal population.

OBJECTIVE: We examined serum in recent postmenopausal women to determine the relationship of menopausal status, as FSH level, to serum acid-base balance. DESIGN, SETTING, AND PATIENTS: Serum electrolytes of 58 women, aged 53-58, were measured relative to serum FSH. The subjects were over one year since the last menstrual period and were from an academic practice setting. RESULTS: In women with FSH <35 IU/L (n=20, mean 16.6 IU/L, SD 6.8), phosphate and lactate were reduced relative to women with FSH >35 IU/L (n=38, mean 84.8 IU/L, SD 34.5). No other major anions showed significant differences. Both groups were analyzed by mass spectroscopy for fatty acids and anionic metabolic intermediates. Lactate was the predominant anion in the organic group but accounted for only about 10% of the FSH-responsive anion change. This change was mainly due to a 0.1-mM increase in phosphate in the high FSH group. CONCLUSIONS: There is a direct correlation of early postmenopausal FSH to increasing serum phosphate. Changes in phosphorus may reflect differences in the rate of bone loss.

A general photonic crystal sensing motif: creatinine in bodily fluids.

We developed a new sensing motif for the detection and quantification of creatinine, which is an important small molecule marker of renal dysfunction. This novel sensor motif is based on our intelligent polymerized crystalline colloidal array (IPCCA) materials, in which a three-dimensional crystalline colloidal array (CCA) of monodisperse, highly charged polystyrene latex particles are polymerized within lightly cross-linked polyacrylamide hydrogels. These composite hydrogels are photonic crystals in which the embedded CCA diffracts visible light and appears intensely colored. Volume phase transitions of the hydrogel cause changes in the CCA lattice spacings which change the diffracted wavelength of light. We functionalized the hydrogel with two coupled recognition modules, a creatinine deiminase (CD) enzyme and a 2-nitrophenol (2NPh) titrating group. Creatinine within the gel is rapidly hydrolyzed by the CD enzyme in a reaction which releases OH(-). This elevates the steady-state pH within the hydrogel as compared to the exterior solution. In response, the 2NPh is deprotonated. The increased solubility of the phenolate species as compared to that of the neutral phenols causes a hydrogel swelling which red-shifts the IPCCA diffraction. This photonic crystal IPCCA senses physiologically relevant creatinine levels, with a detection limit of 6 microM, at physiological pH and salinity. This sensor also determines physiological levels of creatinine in human blood serum samples. This sensing technology platform is quite general. It may be used to fabricate photonic crystal sensors for any species for which there exists an enzyme which catalyzes it to release H(+) or OH(-).

Role of laboratory in the management of phenylbutazone poisoning.

We report a rare case of intentional overdose of phenylbutazone in a 15-yr-old female. The patient exhibited symptoms of phenylbutazone toxicity and the presence of the drug was confirmed by gas chromatography mass-spectrometry (GC-MS) analysis of the initial urine sample. The patient underwent plasmapheresis to remove the drug from the circulation. Semiquantitation of sequential serum samples by GC-MS revealed elimination of phenylbutazone by day 5 of admission at which time the plasmapheresis was discontinued. Elevated blood urea nitrogen (BUN) and creatinine returned to normal. Analysis of biomarkers for liver necrosis and regeneration in sequential serum samples revealed the restoration of normal liver function by day 5. This case further confirms our previous observations that biomarkers for liver necrosis and regeneration can predict the outcome of patients with liver damage due to toxins.

Elevated serum parathormone level after "concise parathyroidectomy" for primary sporadic hyperparathyroidism.

BACKGROUND: Cure after parathyroid exploration is traditionally assessed by serum calcium concentration 6 months postoperatively. Postoperative normocalcemic elevation of serum parathormone (PTH) level has been described but is of unclear significance. METHODS: In a 6-year prospective study of outcomes in 380 patients undergoing initial parathyroidectomy for primary sporadic hyperparathyroidism, we measured intact serum PTH and calcium levels at more than 5 months. Those with normocalcemic high PTH levels were begun on oral calcium + vitamin supplements and monitored. RESULTS: At more than 5 months postoperatively, normocalcemic elevation in serum PTH level occurred in 28% of patients, was more common after resection of double adenomas (P =.01), and predated the onset of recurrent hypercalcemia in 3 of 3 patients with unrecognized multiglandular disease. Although delayed treatment with calcium and vitamin supplements produced no clear benefit, patients who took such supplements from the date of surgery were much less likely to have an elevated serum PTH level more than 5 months later (P =.0005). CONCLUSIONS: After successful parathyroid surgery, compensatory normocalcemic elevation in serum PTH level is frequent and may arise from dietary deficiency. Monitored supplemental intake of calcium and vitamin D appears to prevent or to normalize the condition in most patients. Patients with normocalcemic elevation in serum PTH level should receive evaluation for dietary deficiencies as well as follow-up for possible residual disease.