Preanalytical standardization of sphingosine-1-phosphate, sphinganine-1-phosphate and sphingosine analysis in human plasma by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Preanalytical standardization is required for a reliable quantification of the signaling molecules sphingosine-1-phosphate (S1P), sphinganine-1-phosphate (SA1P) and sphingosine (SPH). METHODS: Methanolic protein precipitation of 15µL EDTA-plasma was applied prior to analysis. Sphingolipids were separated in 3min by hydrophilic interaction liquid chromatography (HILIC, SeQuant™ ZIC®-HILIC column) followed by tandem mass spectrometry. Stability of analytes in whole blood and plasma was investigated. Sphingolipid concentrations were determined in human plasma (n=50) and mice deficient in sphingosine kinase 1 (SK1) and 2 (SK2) (n=5). RESULTS: Storing EDTA whole blood >60min after blood withdrawal at room temperature resulted in an increase in S1P and SPH concentrations of ≥25%. Significant changes in SPH levels of +37% were observed after 60min of storage of EDTA plasma at room temperature. Repeated freeze-thaw cycles of EDTA plasma resulted in increased S1P and SPH levels. Concentrations in human EDTA plasma were between 55.5 and 145.2ng/mL for S1P and between 8.9 and 35.3ng/mL for SA1P. Concentrations of S1P were 36% lower and 96% higher in EDTA-plasma from SK1- and SK2-deficient mice, respectively, compared to the wild type. CONCLUSIONS: Preanalytical standardization is a precondition for the analysis of sphingolipids in human blood.

Ultrasonography for the endocrine surgeon: a valuable clinical tool that enhances diagnostic and therapeutic outcomes.

BACKGROUND: Surgeon-performed ultrasonography (U/S) has revolutionized many subspecialties by broadening the diagnostic and interventional scope of practice. We report our experience on the impact of surgeon-performed U/S in an endocrine surgery practice. METHODS: Prospectively maintained records of patients from November 1999 to November 2004 were reviewed to establish patterns and outcomes of U/S practice. Surgeon-performed neck U/S was done routinely at the initial clinic visit and incorporated into resident/fellow education. RESULTS: A total of 5703 U/S were performed on endocrine patients with thyroid 42%, parathyroid 57%, and adrenal 1% disorders. Diagnostic fine-needle aspiration biopsy (FNA) was achieved with low sampling errors (<7%). When U/S identified thyroid nodules coexisting with hyperparathyroidism, preoperative FNA correctly established benign thyroid diagnosis and minimized need for thyroidectomy. U/S successfully imaged abnormal parathyroid glands when (99)Tc-sestamibi scans were negative. U/S data significantly changed treatment plans in nearly two thirds of thyroid cancer patients. Surgical residents readily mastered essential U/S skills. CONCLUSIONS: Surgeon-performed U/S is a highly specific tool for identification of endocrine disease in the neck. It is learned readily and performed accurately, and functions as an informative extension of physical examination. Because it substantially benefits patient care and impacts surgical decision making, neck U/S is recommended highly as a valuable adjunct to endocrine surgical practice.

Thyrotropin receptor/thyroglobulin messenger ribonucleic acid in peripheral blood and fine-needle aspiration cytology: diagnostic synergy for detecting thyroid cancer.

RT-PCR for thyroglobulin (Tg) and TSH receptor (TSHR) mRNA has been used to detect circulating thyroid cancer cells. Little is known, however, regarding the preoperative sensitivity of this test to detect cancer. Seventy-two patients with thyroid disease (36 with malignancy and 36 with benign disease) were evaluated preoperatively. TSHR and Tg mRNA transcripts were detected by RT-PCR assays, previously determined to be specific for cancer cells. There was 100% concordance between TSHR and Tg mRNA RT-PCR results. Of 36 cancer patients, 11 had recurrent disease, and all were positive by RT-PCR. Among 25 patients with no prior thyroid surgery, 18 tested positive preoperatively (sensitivity 72%). Seven of 36 patients with benign disease tested positive (specificity 80%). The overall preoperative diagnostic accuracy was 77%. Preoperative fine-needle aspiration (FNA) biopsy was performed on 46 of 61 patients with no prior thyroid surgery. FNA was diagnostic in 28 (61%) patients. Preoperative cytology was adequate but not diagnostic in 18 (39%) patients. RT-PCR correctly classified 14 of these 18 patients with indeterminate FNA, and the test detected three of four cancer patients as positive (75% sensitive) and 11 of 14 patients (78% specific) with benign disease as negative. The combined diagnostic performance characteristics for RT-PCR and FNA cytology were sensitivity = 95%, specificity = 83%, and diagnostic accuracy = 89%, with positive and negative predictive values of 84 and 95%, respectively. Our results suggest that the molecular detection of circulating thyroid cancer cells by RT-PCR for TSHR/Tg mRNA complements FNA cytology in the preoperative differentiation of benign from malignant thyroid disease and their combined use may save unnecessary surgeries.

Prospective evaluation of sestamibi scan, ultrasonography, and rapid PTH to predict the success of limited exploration for sporadic primary hyperparathyroidism.

BACKGROUND: The aim of this study was to determine the ability of localizing studies and rapid intraoperative parathyroid hormone (PTH) to predict the success of a limited approach in patients who then underwent bilateral exploration. METHODS: Preoperative sestamibi-iodine subtraction scan and neck ultrasonography (US) were used to direct a focal (1-gland) and unilateral (1-sided) parathyroid exploration by using rapid intraoperative PTH determinations in 350 patients with sporadic primary hyperparathyroidism. Regardless of the findings, the contralateral side was then explored. RESULTS: A single gland was predicted by sestamibi in 290 patients (83%), US in 298 patients (85%), and concordance of both in 205 patients (59%). Unilateral parathyroid exploration, directed by these studies, would correctly identify single-gland disease in only 68%, 74%, and 79%, respectively. The addition of intraoperative PTH would increase the success rate to 73%, 77%, and 82%, respectively. The finding of 2 normal or 2 abnormal glands on 1 side would force bilateral exploration, and additional unsuspected pathology was found in 13%, 13%, and 9%, respectively. This failure rate would increase to 21%, 18%, and 15%, respectively, if the analysis assumed a focal rather than unilateral approach to the initial exploration. CONCLUSIONS: Even in patients with concordant sestamibi and US scans, and an appropriate PTH drop, additional abnormal parathyroid glands were found on complete exploration in 15%. A bilateral approach offers the best opportunity for the long-term cure of primary hyperparathyroidism.

Double adenomas revisited: nonuniform distribution favors enlarged superior parathyroids (fourth pouch disease).

BACKGROUND: A double adenoma (DA) is a recognized clinical entity of primary hyperparathyroidism (HPT) and is presumed to have uniform location distribution. We reviewed our experience with 2-gland parathyroid disease to identify anatomic patterns and implications for surgical management. METHODS: Clinical characteristics were reviewed for 828 consecutive patients with HPT at 2 endocrine referral centers that practice bilateral neck exploration with intraoperative parathyroid hormone (IOPTH) measurement. RESULTS: Fifteen percent (127 of 828) of HPT patients demonstrated 2 enlarged glands; 13% (107 of 828), 3- or 4-gland hyperplasia; and 71% (592 of 828), single adenomas. DAs in superior parathyroids affected 57 of 127 (45%) patients, an observed frequency 3-fold higher than expected (P<.001, chi-square test). DAs were larger than normal glands (240+/-575 mg vs 28+/-23 mg, P<.001), and superior parathyroid adenomas were larger than adenomas at other sites (421+/-983 mg vs 202+/-353 mg, P=.002). Technetium 99 metastable (Tc99m)-sestamibi imaging and IOPTH identified DAs correctly in only 5 of 84 (6%) and 19 of 75 (25%) of patients tested, respectively. CONCLUSIONS: DAs have nonuniform anatomic distribution with predilection for abnormal growth in bilateral superior parathyroids-embryologic remnants of the fourth branchial pouch. Since additional abnormal glands were detected by observation with technetium 99 metastable (Tc99m)-sestamibi imaging and IOPTH rarely aiding detection, unilateral neck exploration may predispose to persistent or recurrent HPT.

Early immunoneutralization of calcitonin precursors attenuates the adverse physiologic response to sepsis in pigs.

OBJECTIVE: The 116 amino acid prohormone procalcitonin and some of its component peptides (collectively termed calcitonin precursors) are important markers and mediators of sepsis. In this study, we sought to evaluate the effect of immunoneutralization of calcitonin precursors on metabolic and physiologic variables of sepsis in a porcine model. DESIGN: A prospective, controlled animal study. SETTING: A university research laboratory. SUBJECTS: 30-kg Yorkshire pigs. INTERVENTIONS: Sepsis was induced in 15 pigs by intraperitoneal instillation of a suspension of cecal content (1 g/kg animal body weight) and a toxinogenic Escherichia coli solution (2 x 10(11) colony-forming units). During induction of sepsis, seven pigs received an intravenous infusion of purified rabbit antiserum, reactive to the aminoterminal portion of porcine prohormone procalcitonin. Another eight control pigs received an intravenous infusion of purified nonreactive rabbit antiserum. For all 15 animals, physiologic data (urine output, core temperature, arterial pressure, heart rate, cardiac index, and stroke volume index) and metabolic data (serum blood urea nitrogen and creatinine, arterial lactate, and pH) were collected or recorded hourly until death at 15 hrs. MEASUREMENTS AND MAIN RESULTS: In this large-animal model of rapidly lethal peritonitis, serum calcitonin precursors were significantly elevated. Amino-prohormone procalcitonin-reactive antiserum administration resulted in a significant improvement or a beneficial trend in a majority of the measured physiologic and metabolic derangements induced by sepsis. Specifically, arterial pressure, cardiac index, stroke volume index, pH, and creatinine were all significantly improved, while urine output and serum lactate had beneficial trends. Treated animals also experienced a statistically significant increase of short-term survival. CONCLUSIONS: These data from a large-animal model with polymicrobial sepsis demonstrate the salutary effect of early immunoneutralization of calcitonin precursors on physiologic and metabolic variables. Immunologic blockade of calcitonin precursors may offer a novel therapeutic approach to human sepsis.