ACTA ENDOCRINOLOGICA (BUC)

Introduction. The determination of phenylalanine (Phe) and tyrosine (Tyr) levels in blood plasma is very important not only in early diagnostic, but also in monitoring the treatment of phenylketonuria (PKU). Purpose. We present a simple, sensitive and accurate procedure to determine simultaneously the plasma concentrations of Phe and Tyr. Procedure. The measurement involves two steps: a) separation of plasma (from blood prelevated on heparin), isolation and preparation of a concentrated solution of amino acids (by ion-exchange column chromatography on Dowex- 50X8), and b) determination of Phe and Tyr concentrations in the solution of amino acids by HPLC (using a Dionex Ultimate 3000 instrument equipped with a diode array detector). The analytical column was a Thermo Scientific Acclaim 120, C18, 5 μm Analitic (4.6 x 250 mm), coupled with an Acclaim C18 guard column. The values of Phe and Tyr concentrations in plasma of several patients were calculated using a calibration curve made with standards of Phe (1834.4 μmol/L in deionized water) and Tyr (600 μmol/L in deionized water). Concentrations as low as 24 μmol/dL of Phe and 15 μmol/dL of Tyr could be determined. Conclusion. The whole procedure presented here is relatively simple, rather inexpensive, however very sensitive and accurate. Consequently, it is very adequate for confirming the diagnosis of PKU in patients with neonatal hyperphenylalaninemia, as well as for monitoring the plasma concentrations of Phe and Tyr in patients with PKU.

Objective. To investigate the impact of body weight on the subclinical hypothyroidism observed in patients with PCOS. Methods. The study included 95 normal weight (Group-1) and 122 overweight or obese women (Group-2) with PCOS. The control group consisted of age and BMI matched healthy individuals and grouped as normal weight (n: 66, Group-3) and overweight or obese (n: 65, Group-4. Women with chronic disease such as overt thyroid dysfunction, late-onset adrenal hyperplasia, and diabetes were excluded from the study. Plasma glucose and lipid profile, thyroid hormones, insulin, FSH, LH, total testosterone, estradiol, progesterone and DHEA-S were measured. Results. While fasting glucose was similar, insulin and HOMA-IR were higher in Group-2 and Group-4 (p: 0.001). The groups were similar with respect to FSH, Estradiol, prolactine, DHEAS. While total testosterone and LH levels were higher (ptestosterone: 0,009), progesterone was lower in both PCOS groups (pprogesterone: 0.041). Free T3, free T4, thyroid antibodies were similar between the groups, but the prevalence of subclinical hypothyroidism was greater in Group-2 and -4 than in Group-1 and -3 (p: 0.044). TSH was only correlated with BMI (r: 0.122, p: 0.02). Conclusion. The increased prevalence of subclinical hypothyroidism in women with PCOS might be the result of increased BMI.

The aim of the study was to evaluate the prevalence of hyperhomocysteinemia in hypothyroid patients and the effect of folic acid supplementation when serum homocysteine\r\n(Hcy) was over risk level.\r\nPatients and methods. Patients with moderate (Group1) and severe hypothyroidism (Group 2) were evaluated before any therapy and after 6 months of combined folic acid and\r\nlevothyroxine substitution, versus control subjects. Hcy, folic acid, thyroid hormones and lipids were measured for all subjects. Thyroglobulin and antithyroglobulin antibodies were measured only for Group 2.\r\nResults. Only 17 % of the cases had basal Hcy at non risk level (<10 mmol/L). Both groups had higher Hcy levels than control (p <0.0001). In Group 1 basal folic acid was lower\r\nthan in control and group 2 (p<0.001). No correlation was found between high levels of Hcy (> 12 mmol/L ) and positive thyroglobulin. After 3 months of combined therapy, significant decrease of Hcy (p<0.0001) was observed compared with the basal level. Normalization of\r\nHcy appears during next 3 months even with reducing the folic acid supplementation.\r\nConclusion. Our results report moderate hyperhomocysteinemia in hypothyroid patients. This may exacerbate the cardiovascular risk traditionally attributed to lipid changes. Six months of combined therapy (L-thyroxine and folic acid) corrected hyperhomocysteinemia excluding the additional risk.

Background: Salivary monitoring of hormone levels has many advantages over the more conventional serum/plasma analysis. Salivary free metanephrines (MN) and normetanephrines (NMN) could precise biochemical diagnosis of pheochromocytoma (PHEO) as an alternative to plasma metabolites.\r\nSubjects and methods: The prospective case-control study included a group of 30 patients confirmed with PHEO an age-matched control group of 70 normotensive subjects. The PHEO diagnosis was suspected on clinical ground and confirmed by imaging studies and classical neuroendocrine markers. Free plasma and salivary NMN and MN were assayed using enzyme immunoassay for both metabolites.\r\nResults: In tumor cases all metabolites were increased. As expected, values for all 4 parameters (mean?SEM) differed significantly in tumor group vs. normal group: free plasma\r\nnormetanephrines (NMNp): 1514.16 ? 282.97 pg/mL vs 47.82?2.52 pg/mL; free salivary normetanephrines (NMNs):\r\n663.63?168.47 pg/mL vs 44.98? 2.47 pg/mL; free plasma metanephrines (MNp): 445.20 ? 99.92 pg/mL vs 18.87?1.03\r\npg/mL; free salivary metanephrines (MNs):206.60?91.48 pg/mL vs 14.47?0.72 pg/mL with significant correlations in all\r\n100 subjects. Passing & Bablok regression showed no significant deviation from linearity in Elisa assay of NMNs vs NMNp; a significant deviation from linearity existed\r\nin Elisa assay of MNs vs MNp. Cut-off values, sensitivity and specificity for all 4 parameters were calculated by ROC\r\nanalysis. Plasma and salivary normetanephrines proved similar sensitivity (100%) and specificity (100%). Pairwise\r\ncomparison of ROC curves areas showed no significant differences between NMNp vs NMNs and MNp vs MNs. Ten cases were investigated post-surgery. All 4 parameters\r\nshowed no significant differences vs. control group.\r\nConclusions: Salivary free normetanephrines could be used as a nonstressful marker for diagnosis purpose in pheochromocytoma proving similar sensitivity and specificity as plasma free normetanephrines.

Background. Probiotic fermented milk is one of the most beneficial foods. The main purpose of this study was to investigate the effect of probiotic fermented milk on the serum level of insulin and homocysteine in the type 2 diabetes patients. Methods. This study was done in 60 patients with type 2 diabetes. The intervention group received 600 mL of probiotic fermented milk (kefir) daily and control group received 600 mL of conventional fermented milk daily for 8 weeks. Food intake, anthropometric indices, serum parameters were assessed at the beginning and at the end of the study. The statistical analysis was done by the use of SPSS software (Ver.13). Results. The mean of serum insulin level did not reduce significantly after the intervention in probiotic fermented milk group, and there was no significant difference between the two groups. The mean of HOMA-IR decreased significantly in probiotic fermented milk group after intervention and there was a significant difference between the two groups after intervention. The mean of quickie increased in probiotic fermented milk group, but this increase was not significant. Also, there was not significant difference between the two groups after intervention. The mean of homocysteine level decresead significantly in patients with probiotic fermented milk and conventional fermented milk consumption. Conclusions. By considering the effect of probiotic fermented milk on some risk factors of cardiovascular disease in diabetic patients, probiotic foods may be useful as an adjuvant therapy in diabetic patients.

Background. An increasing number of studies suggest that hypothyroidism may lead to hepatorenal toxicity. This study examined whether thymoquinone (TQ), the main active Nigella sativa constituent, could prevent the detrimental influences of hepatorenal toxicity of hypothyroidism during the juvenile period in rats. Methods. The male rats were randomly divided into four groups (n = 7), including control, propylthiouracil (PTU), PTU-TQ 5 mg/kg, and PTU-TQ 10 mg/kg. PTU was dissolved in drinking water at a concentration of 0.05% and administered for six weeks. In the PTU-TQ5 and PTU-TQ10 groups, animals received PTU plus 5 mg/kg and 10 mg/kg of the TQ (i.p.) for six weeks, respectively. The rats were evaluated after TQ treatment by measuring serum markers of liver and kidney function tests as well as oxidative stress biomarkers in liver and kidney tissues. Results. Administration of TQ (5 and 10 mg/ kg) decreased oxidative stress damage in liver and kidney tissue in hypothyroidism rats with improvement in activities of antioxidant enzymes and a decrease in MDA in both liver and kidney homogenates. Furthermore, TQ treatment significantly inhibited the elevation of serum biochemical markers of liver and kidney function associated with this hepatorenal toxicity. Conclusion. These results suggest that the protective effect of TQ in hypothyroidism-induced hepatorenal toxicity in rats is attributed to its ability to reduce oxidative stress in hepatic and renal tissues. However, more studies are recommended to investigate the exact mechanism (s) for the effect of TQ on hepatorenal outcomes of hypothyroidism in human subjects.

Background. Although cardiovascular risk is increased in patients with subclinical hypothyroidism (SCH), replacement therapy is not recommended in those with TSH levels\r\nbetween 5 and 10 mU/L.\r\nObjective. We aimed to evaluate the effects of levothyroxine (LT4) treatment on cardiovascular risk factors and carotid artery intima media thickness (CIMT) in patients with SCH who had TSH levels between 5 and 10 mU/L.\r\nSubjects and Methods. Sixty SCH patients with TSH levels between 5 and 10 mU/L were included in the study. Patients\r\nwere randomized into two groups as treatment (n=30) and control (n=30) groups. BMI, blood pressure, lipid profile, fibrinogen, homocysteine, hs-CRP and CIMT were measured in all patients at baseline and after six months. LT4 treatment was initiated and the dose was tapered according to TSH levels in treatment.\r\nResults. There was no significant difference between baseline and six month measurements in the control group. However, TSH, LDL-C, fibrinogen and mean CIMT measurements were decreased and HDL-C level was increased in the treatment group.\r\nConclusions. We suggest that LT4 therapy is necessary for the prevention of modifiable cardiovascular risk factors in\r\npatients with TSH levels between 5 and 10 mU/L.

Dual X-ray absorptiometry (DXA) is well known as the “gold standard” technique for the diagnosis of osteoporosis; therefore its “brightness” puts into shadow other valuable applications both in research and clinical practice. Whole body scan offers the opportunity to analyze the body composition, the oldest non bone densitometric application with valuable research data relevant for sarcopenia diagnosis, obesity and lipodystrophy diagnosis. Hip geometry analysis is also an older feature of DXA femur scan remained in the research area. The trabecular bone score (TBS) is the newest and most astonishing application using DXA scans: it brings bone quality data based on the image texture analysis of the spine DXA images with relevant impact on the fracture risk assessment independent of the bone density. Vertebral fracture assessment could be done in the same visit with bone densitometry using DXA rapid lateral spine scan: it is a borrowed application from plain radiology, as atypical femoral fracture (AFF) diagnosis and relies on the increased image quality of the DXA scans on recent equipments. At the same time, incidental findings on these images offer the opportunity to evaluate aortic calcifications useful for cardiovascular risk evaluation.

Background. Saturated free fatty acids, such as palmitate, can cause pancreatic β-cell apoptosis. Although the toxicity of palmitate could be mediated partly through endoplasmic reticulum (ER) stress, the mechanism by which fatty acid over-accumulation led to lipoapoptosis in β-cells has not been fully understood. Recently, the fat mass and obesity associated (FTO) gene is proved to be related to obesity and type 2 diabetes, but its function in β-cells remains largely unknown. Whether or not FTO could counteract palmitate induced β-cell apoptosis remains to be investigated. Methods. INS-1 cells were infected with FTO expression adenovirus and incubated with palmitate. Then, viability and induction of apoptosis were measured by MTT assay and Hoechst-staining, respectively. Western blot analyses were performed for unfolded protein response specific proteins and mRNA expression of target molecules was determined by real time-PCR. Results. Palmitate incubation led to β-cell apoptosis, whereas adenovirus-mediated FTO overexpression significantly ameliorated the effect of palmitate. Increased activation of X-box binding protein 1 (Xbp1) mRNA and phosphorylation of eIF2α were also observed after palmitate treatment, whereas FTO overexpression significantly ameliorated the effect of palmitate. The proapoptotic transcription factor CHOP was significantly enhanced by palmitate incubation. In contrast, in accordance with sustained cell survival, FTO overexpression resulted in notably decreased CHOP levels. Interestingly, mRNA expression of the chaperones Pdi, Calnexin and Grp94 was not altered by palmitate treatment, while FTO overexpression notably increased the expression of Bip. Conclusion. Our data showed that FTO overexpression could protect β-cells from palmitate-induced apoptosis partly through suppression of ER stress.

Background. We aimed to evaluate whether a high carbohydrate or a high fat diet differs in alteration of the inflammatory and metabolic risk factors in cardio-renal metabolic syndrome in rats. Methods. Twelve male Wister rats were randomly divided into two groups: one received diet 1 standard pellet rat diet (D1) containing 10% fat, 50% carbohydrate, 25% protein and another group received diet 2 (D2) containing 59% fat, 30% carbohydrate and 11% protein for 16 weeks. Weight was recorded weekly. FSG and insulin levels were measured using an enzymatic spectrophotometric and a standard ELISA kit respectively. Inflammatory parameters including TGF-β, MCP-1, TNF-α, IL-1β, IL-6 in the renal and cardiac tissues of rats were evaluated by ELISA technique. Result. Food intake in D1 and D2 groups increased in the study period, however food intake in D2 group was significantly higher compared with D1 group. FSG, HOMA and TG concentrations in D2 group were significantly higher compared to D1 group. Moreover, TGF-β and MCP- 1 concentrations in the renal tissues of D2 group and TNF-α in the cardiac tissues of D1 group were significantly higher compared with D1 group (P<0.05). Positive associations between IL-1β and TG and between HOMA, FSG with TGF-β and MCP-1 in the renal tissue of animals were also identified.