Screening of the Men1 gene and discovery of germ-line and somatic mutations in apparently sporadic parathyroid tumors.

Hyperparathyroidism is the first manifestation in a majority of multiple endocrine neoplasia (MEN1) patients. To discriminate between sporadic and hereditary parathyroid tumors and characterize MEN1 somatic mutations, we examined MEN1 gene mutations in patients who had undergone surgery for sporadic parathyroid tumors. DNA was extracted from fresh frozen parathyroid tumor specimens from 112 patients as well as from peripheral blood leukocytes from 64 of the 112 patients. Sequence analysis was performed to examine exons 2-10 of the MEN1 gene for mutations. Loss of heterozygosity (LOH) was also examined by an analysis of codon 418 and 541, which lie within a polymorphic region of MEN1. Somatic MEN1 mutations were found in 25 of the 112 patients (22%). Two patients had two point mutations (508del33 and Y341X and 363insT and 1767delT, respectively). A total of 27 mutations were characterized, 20 of which have not been reported previously. There were 7 nonsense mutations, 10 frameshift mutations, 2 splice site deletions, 5 missense mutations, and 3 in-frame mutations. Nineteen mutations (70%) predicted truncation of the menin protein. Germ-line MEN1 mutations were found in 3 of 64 patients (5%) who had no family history of endocrine tumors associated with MEN1, and these patients were identified as MEN1 gene probands. LOH at the MEN1 locus was detected in three parathyroid tumors showing germ-line mutation. LOH was significantly frequent in parathyroid tumors with somatic MEN1 mutations (15 of 22 tumors, 68%) but not in those without germ-line or somatic MEN1 mutations (14 of 51 tumors, 28%; P = 0.0011). Our findings suggest that alterations of both alleles of the MEN1 gene may be associated not only with endocrine tumors of affected MEN1 patients but also with sporadic parathyroid tumors. Germ-line MEN1 gene analysis can distinguish heritable from nonheritable parathyroid tumors, and MEN1 gene evaluation of patients with apparently sporadic parathyroid tumor is recommended before parathyroid surgery.

Advanced glycation end products of the Maillard reaction in aortic pepsin-insoluble and pepsin-soluble collagen from diabetic rats.

Recent immunohistological studies using antibodies against advanced glycation end products (AGEs) have demonstrated the presence of AGEs in several tissues. By an enzyme-linked immunosorbent assay using the monoclonal anti-AGE antibody, the present study aimed to determine AGEs in pepsin-insoluble collagen (PIC) as well as in pepsin-soluble collagen (PSC) from the aortas of streptozotocin (STZ)-induced diabetic rats (at 4, 16, and 28 weeks after STZ injection) and those of age-matched control rats. Addition of EDTA to the immunoassay buffer has led us to successful determination of AGEs in the aortic PIC samples with following results: 1) in diabetic rats, there was a time-related increase in the AGE contents at 28 weeks (n = 9, 226.4 +/- 13.5 ng/mg collagen [mean +/- SE]), compared with that at 4 and 16 weeks (n = 6, 79.6 +/- 9.5 ng/mg collagen, and n = 8, 149.4 +/- 30.9 ng/mg collagen at 4 and 16 weeks, respectively; both P < 0.05, between 4 and 16 weeks and 28 weeks); 2) after 28 weeks of diabetes, the AGE contents in PIC of aortas were significantly higher in diabetic rats than in controls (n = 9, 226.4 +/- 13.5 ng/mg collagen vs. n = 8, 129.6 +/- 14.9 ng/mg collagen, P < 0.01, diabetic vs. control); and 3) the level of the AGE content was strongly correlated with the PIC/total collagen (TC) ratio (n = 45, r = 0.698, P = 0.0001). By treating the samples of PSC with alkaline solution, the AGE content of PSC was also determined. In the PSC fraction, the AGE levels in the diabetic rats tended to increase with time and to be higher than those of control rats at 28 weeks although these changes were not statistically significant (diabetic: n = 4, 19.4 +/- 9.7; n = 6, 22.3 +/- 6.2; n = 6, 39.6 +/- 10.8; control: n = 4, 19.7 +/- 9.8; n = 6, 22.9 +/- 7.3; n = 7, 30.7 +/- 7.2; at 4, 16, and 28 weeks, respectively). Compared with the AGE levels of PSC, those of PIC were about four to seven times and four to five times higher in diabetic and control rats, respectively (PIC versus PSC in diabetic or control rats, all P < 0.001, at 4, 16, and 28 weeks, respectively). These findings provide the first immunochemical evidence that AGE adducts are present in the materials extracted sequentially by pepsin and collagenase and that these adducts in PIC accumulated as a function of the increase in the aortic PIC/TC ratio.

Glycoxidation in aortic collagen from STZ-induced diabetic rats and its relevance to vascular damage.

Glycoxidation reactions lead to the formation of permanent, irreversible chemical modifications and cross-links in protein, such as the glycoxidation products carboxymethyllysine (CML) and pentosidine. It has been implicated that CML as well as Amadori products play a role in the formation of superoxidative products, such as H2O2 and advanced glycosylation endproducts in trapping LDL. Therefore, a possible relationship between glycoxidation and lipoperoxidation might exist because oxidized lipoprotein, which has been directly linked to atheroma formation, could be produced by the superoxidative products released from the pathway of CML formation. Using a CML-specific monoclonal antibody (6D12) and a specific antiserum against hexitol-lysine (HL), an Amadori product, we studied the relationship between glycoxidation and lipoperoxidation by determining the aortic CML contents with ELISA and the fluorescence levels of lipoperoxidation side products, malondialdehyde (MDA) and hydroxynonenal (HNE) from STZ-induced diabetic rats and age-matched control rats. The immunohistochemical and ultrastructural changes relevant to glycoxidation and lipoperoxidation were also studied. The CML content measured by ELISA in DM rats was significantly higher than that in the control rats at 28 weeks (n = 11, P < 0.01). The levels of MDA-linked and HNE-linked fluorescence in the DM rats increased in a similar way and were significantly higher than the levels in control rats at 28 weeks (n = 11, both P < 0.01 at 28 weeks). The CML contents correlated with the fluorescence levels of both MDA-linked (n = 19, r = 0.638, P < 0.01) and HNE-linked fluorescence (n = 19, r = 0.629, P < 0.01) only in the DM rats, but not in the control rats. Our immunohistochemical study thus demonstrated that CML was initially formed in the aortic media of diabetic rats in the 16th week of diabetes, localized primarily in the extracellular matrix surrounding the aortic smooth muscle cells after HL occurred early in the 2nd week of diabetes. Consequently, a significant increase in the extracellular matrix and decrease in the area of the SMCs were observed in the aortic media in the DM rats by a morphometrical study. The in vivo results of this study provided the first evidence that CML correlated with fluorescence levels of MDA and HNE, and thus suggested the existence of a close relationship between glycoxidation and lipoperoxidation in vivo. This information is thus considered to shed some new light on the etiology of atherogenesis in diabetes.

Usefulness of quick intraoperative measurements of intact parathyroid hormone in the surgical management of hyperparathyroidism.

We investigated the use of quick measurement of intraoperative intact parathyroid hormone (I-PTH) to predict the outcome of parathyroidectomy. We examined intraoperative monitoring of I-PTH in 34 consecutive primary hyperparathyroidism (pHPT) patients operated on between April and December 1999. The average patient age was 56 +/- 13 years, and all but one were women. Four had a history of thyroidectomy. Blood samples were drawn before excision of enlarged parathyroid gland(s) and at 2, 5, 10, and 15 minutes afterward. Plasma I-PTH was measured by a two-site immunochemiluminometric assay. Twenty-three patients were shown to have single gland disease, and ten had multiglandular disease. All patients, except one, underwent successful parathyroidectomies. The plasma I-PTH value 15 minutes after removal of enlarged gland(s) had dropped to 26 +/- 10% of pre-excision I-PTH value. In one patient with a previous history of thyroidectomy for thyroid papillary cancer, no gland enlargement was found in the area where the lesion had been suggested by both ultrasonography and 99mTc sestamibi scanning. In this case, intraoperative measurements of I-PTH in the bilateral internal jugular veins identified an ectopic parathyroid tumor, which was successfully removed. We conclude that quick measurement of intraoperative I-PTH is a valuable tool for decision-making, especially for reoperative parathyroid surgery, for patients with previous history of thyroidectomy, and for patients in whom unilateral neck exploration or a single-gland approach is scheduled based upon preoperative localization.

Evaluation of recurrence in 36 subacute thyroiditis patients managed with prednisolone.

OBJECTIVE: The incidence of subacute thyroiditis (SAT) is low and there are a few reports of recurrence of subacute thyroiditis. Current treatment protocols for SAT are not uniform. Prednisolone (PSL) is chosen more often for treatment than nonsteroidal anti-inflammatory drugs. This study was undertaken to confirm the recurrence rate of SAT managed by PSL, and to compare the initial laboratory data between the recurrent and the non-recurrent groups. METHODS: After diagnosis, all patients were treated with PSL (starting at 30 mg or 25 mg per day, tapered by 5 mg per week) for 5 or 6 weeks. We evaluated data and symptoms at the first visit and during the therapy. PATIENTS: Thirty-six patients who received only PSL for SAT at our hospital between January 1997 and December 1998 were referred. These patients asked to visit every 2 weeks for the monitoring of symptoms and laboratory data. RESULTS: SAT symptoms recurred in eight patients (22%), most upon cessation of PSL. There was no difference in initial serum sialic acid, erythrocyte sedimentation rate, C-reactive protein, thyroglobulin, serum free thyroxine and free triiodothyronine before PSL treatment between the recurrent and non-recurrent patient populations. CONCLUSIONS: The recurrence rate of SAT with treated PSL is about 20%. There was no difference in the laboratory data before starting the therapy between recurrent and non-recurrent groups. Therefore, a modified protocol of PSL administration may be needed to decrease the early recurrent rate of SAT.

Thin-layer chromatography and high-performance liquid chromatography for the assay of fatty acid compositions of individual phospholipids in platelets from non-insulin-dependent diabetes mellitus patients: effect of eicosapentaenoic acid ethyl ester administration.

Eight major phospholipids were separated by a TLC method with a one-dimensional developing system without any pretreatment of the plate and the fatty acids incorporated into each phospholipid class were analysed by an improved HPLC method with a simple elution system, which has advantages with respect to resolution and analysis time. The fatty acid compositions of individual phospholipids in platelets were investigated following administration of ethyl cis-5,8,11,14,17-eicosapentaenoate for more than 13 weeks to patients with non-insulin-dependent diabetes mellitus. The cis-5,8,11,14,17-eicosapentaenoic acid compositions of all phospholipid classes were significantly increased with decreasing platelet aggregation rates after the administration. These results suggested that the present method provides the complete separation of individual phospholipids in sufficient amounts to allow fatty acid analysis on the isolated phospholipid moieties.

High-performance liquid chromatographic measurements of urinary hydroxycarboxylic acids as an index of the metabolic control in non-insulin-dependent diabetic patients.

Hydroxycarboxylic acids in urine of patients with non-insulin-dependent diabetes mellitus and of healthy subjects are analyzed as 2-nitrophenylhydrazides by an improved high-performance liquid chromatographic method which has advantages with respect to resolution and analysis time. Variations in levels of hydroxycarboxylic acids, originated from the metabolism of valine, leucine and isoleucine, have been described in the diabetic patients who have good and poor metabolic controls. The sum of the hydroxycarboxylic acids in both groups of diabetic patients was significantly increased compared with the values of the healthy subjects. Statistically significant difference was present between the two groups. In the whole group of diabetic patients, the sum of the hydroxycarboxylic acids correlated with fasting plasma glucose or hemoglobin A1c (r = 0.548, P < 0.01 and r = 0.629, P < 0.01, respectively). These results suggest that the relevance of these abnormalities may be used as an index of metabolic control in diabetic patients.