Simultaneous estimation of total homocysteine and methylmalonic-acid using LC-MS/MS: Clinical application in adult and pediatric.

Quantitative determination of biomarkers homocysteine (Hcy) and methylmalonic acid (MMA), the regulators of cobalamin (Cbl) and folate levels, together used as a biomarkers to diagnose chemical insufficiency/deficiency of Cbl and folate. We report simultaneous clinical estimation of total Hcy and MMA with efficient clean-up, sensitive and selective LC-MS/MS method. Efficient sample clean-up was achieved by a two-step extraction protocol with 100 µL serum. The validated method was applied to 893 clinical samples from 2 cohorts including pediatrics and mothers, respectively, for identifying their Cbl and folate status. The method shows excellent order of linearity for Hcy (22.2nM-3.7 µM) and MMA (42.34 nM - 5.92 µM), respectively. Complete method validation was performed where intraday-interday accuracy-precision and mean stability recovery data were found within ±15%. The validated method was extended for the quantification of serum total Hcy-MMA levels in clinical samples. The efficient extraction with negligible matrix-effect (ME) has reduced LC-MS/MS chocking and clean-up downtime. The rapid, sensitive and robust LC-MS/MS method has been successfully validated for simultaneous estimation of total Hcy and MMA using only 100 µL serum. The method was applicable to large number of clinical samples and was found to be good throughput with low contamination of mass detector, high sensitivity and selectivity.

Multielemental Analysis and In Vitro Evaluation of Free Radical Scavenging Activity of Natural Phytopigments by ICP-OES and HPTLC.

The phytopigments derived from ethnomedicinal plants employed as traditional medicines appear to be the simplest alternative for artificial radical colorants. This can be because of persistent use of synthetic dyes and their harmful impacts linked to human lives as well as to the ecosystem. The literature evidences clearly reveal the complications from growing demands of radical colorants from artificial origin. The planned analysis work hence focuses on screening of the fundamental composition of phytopigments, obtained from plant sources by subtle technique of ICP-OES, with axial plasma combined with nebulizer motor-assisted gas flow approach, utilizing microwave digester for complete digestion of phytopigments, thereby establishing the pigments being safe for consumption. Additionally, the observations from free radical scavenging activity using DPPH by HPTLC concluded that the natural pigments obtained from plant sources are rich in flavonoids with potent antioxidant property. Thus, an effort has been made through the developed ICP-OES methodology, to beat the distinct imprecise practice of food labeling, once natural pigments are utilized in a variety of additives, as food colorants with amounts of components detected as arsenic, lead, and metal, within specified limits of FSSAI, demonstrate and establish safety of natural foodstuff agents, as compared over hazardous synthetic azo dyes.

Prolonged fasting reduces rat hepatic beta 1 thyroid hormone receptor protein without changing the level of its messenger ribonucleic acid.

The level of hepatic nuclear T3-binding capacity falls in rats subjected to fasting. To define the mechanism underlying these changes, we have assayed in liver the concentration of the mRNA coding for the beta 1-receptor (beta 1-TR) isoform, the total nuclear T3-binding capacity, and the fraction of the total binding capacity that can be specifically immunoprecipitated with an anti-beta 1-TR immunoglobulin G preparation. Although no changes in beta 1-TR mRNA concentration were noted, we observed a 60% fall in total binding capacity. beta 1-TR mRNA levels were preserved despite a 50% fall in total poly(A)+ RNA. The fall in beta 1-TR protein, however, was consistent with a generalized decrease in total hepatic protein content. This study provides yet another instance in which measurement of receptor mRNA is not consonant with the behavior of the nuclear T3 receptor protein.

5'-Monodeiodinase activity in developing human cerebral cortex.

Iodine status of aborted fetuses (11-25 wk gestation) was determined on the basis of maternal thyroid status and urinary iodine excretion. Fetal cerebral cortex thyroxin and 3,5,3'-triiodothyronine (T3) contents peaked at 15-18 wk gestation and then fell in iodine sufficiency. In mild iodine deficiency T3 concentration was maintained at a higher level until week 22, although it were still significantly less than in the iodine-sufficient group. Reverse T3 (rT3) rose from 11 to 22 wk gestation without any effect of iodine status. The activity of 5' and 5-monodeiodinase also increased from 11 to 22 wk gestation, with 5' monodeiodinase activity significantly increased by mild and moderate iodine deficiency. In contrast, cerebral cortex 5-monodeiodinase activity was significantly lower in moderate iodine deficiency at 15-18 wk gestation. The observations indicate that cerebral cortex 5' and 5 monodeiodinase activities are modulated in iodine deficiency to enhance T3 production from thyroxin (T4) during the period that coincides with neuroblastogenesis.

Learning disabilities and poor motivation to achieve due to prolonged iodine deficiency.

The effect of prolonged iodine deficiency on learning and motivation was studied. One hundred male children--matched for age, socioeconomic status, and formal education--were selected from both severely iodine-deficient (SID) and mildly iodine-deficient (MID) villages. Mean urinary iodine excretion was significantly lower in the SID than in the MID group (219.84 +/- 57.52 compared with 449.14 +/- 32.31 nmol/L, P < 0.001). The serum thyroxine concentration was significantly lower (90.36 +/- 6.46 compared with 123.70 +/- 15.42 nmol/L, P< 0.001) and serum thyroid-stimulating hormone (TSH) was significantly higher in the SID group than in the MID group (6.23 +/- 0.34 compared with 4.85 +/- 0.28 mU/L, P< 0.01). The children were administered maze, verbal, and pictorial learning tasks and a test of motivation. The results showed that SID children are slow learners compared with MID children. In both groups the rate of learning over trials was superior in younger (aged 9-12 y) children although the initial performance of older (aged 12-15 y) children was better (P < 0.01). SID children scored significantly lower than MID children on the achievement motivation scale (P < 0.01). The results are suggestive of neural impairment as well as poor sociopsychologic stimulation, resulting in learning disability and lowered achievement motivation. Unless iodine nutrition is improved in the community as a whole, these abnormalities may prevent millions of children from the SID areas from achieving their full potential even if learning opportunities are made available to them.

Hypothyroidism alters mitochondrial morphology and induces release of apoptogenic proteins during rat cerebellar development.

Thyroid hormone (TH) deficiency leads to extensive apoptosis during cerebellar development, but the mechanism still remains unclear. Different signals also converge on mitochondria during apoptosis to induce the release of apoptogenic proteins that activate proteolytic cascade through specific enzymes called caspases. Here we studied the effect of hypothyroidism on alterations in mitochondrial structure and translocation of apoptogenic molecules during rat cerebellar development. Structural analysis of mitochondria was studied by electron microscopy. The translocation of apoptogenic molecules was analyzed by Western blotting. TH deficiency led to vacuolization, enlargement and decrease in the number of cristae. The majority of the proapoptotic molecule, Bax, was localized in mitochondria under hypothyroid conditions whereas a limited presence of Bax was detected in the euthyroid state. Translocation of cytochrome c, apoptosis-inducing factor (AIF) and second mitochondrial-derived activator of caspases (SMAC) from mitochondria to cytosol was detected primarily in early developmental stages in the hypothyroid condition. These experimental results demonstrate that TH maintains mitochondrial architecture and inhibits the release of apoptogenic molecules to prevent excess apoptosis during cerebellar development.

Hypothyroidism alters the expression of Bcl-2 family genes to induce enhanced apoptosis in the developing cerebellum.

Thyroid hormone (TH) deficiency results in delayed proliferation and migration of cerebellar granule cells. Although extensive cell loss during the development of the cerebellum under hypothyroid conditions is known, its nature and its mechanism are poorly understood. Bcl-2 family gene expression is known to determine the fate of cells to undergo apoptosis. We evaluated the effect of hypothyroidism on Bcl-2 family gene expression in the developing rat cerebellum. Electrophoresis and Western blotting were used to analyze DNA fragmentation and expression of DNA fragmentation factor (DFF-45), Bcl-2, Bcl-xL and Bax genes respectively. In the hypothyroid condition, extensive DNA fragmentation and enhanced cleavage of DFF-45 were seen throughout development (postnatal day 0 to day 24) and adulthood whereas they were absent in the euthyroid state. The anti-apoptotic genes Bcl-2 and Bcl-xL were down-regulated and the pro-apoptotic gene Bax was expressed at higher levels compared with the euthyroid state. These results suggest that normal levels of TH prevent cerebellar apoptosis to a large extent, whereas hypothyroidism not only increases the extent but also the duration of apoptosis by down-regulating the anti-apoptotic genes and maintaining a high level of the pro-apoptotic gene Bax.

Comparative study of ELISA and indirect immunofluorescence for the detection of anti-neutrophil cytoplasmic antibodies. Evaluation of the SCIMEDX/EURO Diagnostica ELISA assay in a clinical setting.

This study evaluates the performance of a commercial ELISA assay for detection of anti-neutrophil cytoplasmic antibodies (ANCA). Anti-proteinase 3 (anti-PR3) and anti-myeloperoxidase (anti-MPO) ELISA by (SCIMEDX/EURO Diagnostica, Denville, NJ) were compared with the indirect immunofluorescence (IIF) method using ethanol-fixed neutrophils. Three hundred sixty serum samples were examined. There was complete correlation of results in 357 (99.2%) cases between cANCA pattern and anti-PR3 results, and in 356 (98.9%) cases between pANCA pattern and anti-MPO ELISA results. The specificity of ELISA for detection of vasculitis was 95.7% and sensitivity was 60%, whereas for IIF, they were 95.7% and 65%, respectively. The ELISA showed good reproducibility with interassay variation (CV), ranging from 7.1% to 13.2%. Our results show that SCIMEDX/EURO Diagnostica ELISA for ANCA is a reliable and reproducible method that is as good as IIF for detection of ANCA.

Organochlorine pesticide exposure and thyroid function: a study in human subjects.

We examined the serum levels of thyroxine and thyroid stimulating hormone in 103 rural subjects with respect to blood levels of organochlorine pesticide and occupation. We found that 24.3% of study subjects had depleted thyroxine levels in association with significantly lower organochlorine pesticide residues in blood. Sex, nutritional status, thyromegaly, or handling of pesticides in the course of work were not found to be factors contributing to depleted thyroxine levels.

Iopanoic acid as an adjunct to carbimazole in the management of hyperthyroidism.

BACKGROUND: The thiourea drugs take a few weeks to control the symptoms of hyperthyroidism whilst iodine containing radiographic contrast agents (iopanoic acid and sodium ipodate) have a more rapid effect. There is no report on the use of iopanoic acid administered in conjunction with carbimazole, so we evaluated the efficacy of this combination in the early medical management of patients with hyperthyroidism. METHODS: Thirty hyperthyroid patients diagnosed by clinical and biochemical criteria were randomized into two treatment groups. Group A (n = 16) received iopanoic acid (500 mg orally twice a day for the first 3 weeks) and carbimazole (30 mg orally in three divided doses) while group B (n = 14) received carbimazole alone. Clinical examination and estimation of serum total T3, total T4 and TSH were done by radioimmunoassay at the start of therapy, weekly for 4 weeks and then at 6, 8 and 12 weeks. RESULTS: In the initial 3 weeks, iopanoic acid induced a significantly greater fall in mean serum total T3 levels (Z = 2.298, p < 0.02) and a slower fall in mean serum total T4 (Z = 2.396, p < 0.05) in group A patients compared to those in group B. This was accompanied by earlier clinical improvement in group A patients. The mean serum total T3 and T4 values rose to higher levels in group A at 4 weeks, one week after discontinuation of iopanoic acid. At the end of 12 weeks, however, there was no significant difference in the mean serum total T3 and T4 levels between the two groups (p > 0.05). Biochemical euthyroidism (i.e. total T3 < 3 nmol/L and total T4 < 170 nmol/L) was achieved later in group A patients than in group B (10.4 +/- 5.0 weeks v. 3.6 +/- 1.2 weeks, p < 0.0001). CONCLUSIONS: Iopanoic acid given together with carbimazole induces rapid clinical improvement in hyperthyroid patients than carbimazole alone. However, the delayed achievement of euthyroidism may preclude its routine use in the management of patients with hyperthyroidism except in those with thyrotoxic emergencies.

Single daily dose of carbimazole in the treatment of hyperthyroidism.

BACKGROUND: The antithyroid drugs, methimazole and carbimazole, are conventionally used in divided daily doses. However, these drugs have a longer intrathyroidal than a plasma half-life. We undertook this prospective, controlled study, in an area of mild iodine deficiency, to compare the efficacy of a single daily dose of carbimazole with divided doses in the treatment of hyperthyroidism. METHODS: Nineteen patients with hyperthyroidism received 30 mg of carbimazole daily at bed time (group A) while 14 received 10 mg of carbimazole every 8 hours (group B). These patients were assessed clinically and biochemically by estimation of serum total thyroxine, total triiodothyronine and thyrotropin before and 1, 2, 3, 4 and 6 weeks after treatment. RESULTS: There was no significant difference between mean baseline concentrations of thyroxine and triiodothyronine. After 1, 2, 3, 4 and 6 weeks there was a decline in their concentrations which was similar in both groups (p > 0.05). Euthyroidism was achieved in 4.6 +/- 1.4 weeks (range 2-6 weeks) in group A and in 3.8 +/- 1.2 weeks (range 3-6 weeks) in group B (p > 0.05). CONCLUSIONS: We conclude that carbimazole in a single daily dose is an effective method for treating hyperthyroidism in an area of mild iodine deficiency and its efficacy is comparable to divided dose therapy. This practical and acceptable method of treatment can be specially useful in patients who find it difficult to remember to take divided doses.

Extraovarian endocrine abnormalities in north Indian women with premature ovarian failure.

BACKGROUND: Premature ovarian failure is a rare syndrome characterized by cessation of menstruation before 35 years of age associated with an elevated gonadotropin level (serum follicle stimulating hormone > 40 IU/L) and oestrogen deficiency. Premature ovarian failure is often the result of an autoimmune process and involvement of other endocrine glands has been reported but available studies are hampered by a lack of uniformity in diagnostic criteria. METHODS: The endocrine profile of 37 north Indian women with premature ovarian failure was studied. The investigations included measurement of serum follicle stimulating hormone, luteinizing hormone, oestradiol, prolactin, total thyroxine, total triiodothyronine and thyroid stimulating hormone. To assess adrenocortical reserve, adrenocorticotropic hormone stimulated plasma cortisol levels were estimated. RESULTS: Eighteen (49%) patients had extraovarian endocrine abnormalities. Eight (22%) had abnormal thyroid function tests and 12 of 29 (41%) patients tested for adrenocortical reserve showed impaired response of plasma cortisol to adrenocorticotropic hormone stimulation. CONCLUSION: Extraovarian endocrine (especially adrenocortical) involvement is frequent in patients with premature ovarian failure. Because none of the patients had overt clinical evidence of thyroid or adrenocortical disease, we suggest that tests for thyroid and adrenal function be performed routinely in females presenting with premature ovarian failure.

Association of common carotid intima-media thickness and lipoprotein(a) with coronary artery disease.

BACKGROUND: Carotid artery intimal medial thickness is a simple, non-invasive and reproducible clinical tool to evaluate atherosclerosis and predict coronary artery disease. Lipoprotein(a) levels are related to both atherogenesis and thrombogenesis and may be a key link between lipid and coronary artery disease. This study evaluated the association of carotid intimal medial thickness and lipoprotein(a) with coronary artery disease. METHODS AND RESULTS: We studied 185 randomly selected patients hospitalized for coronary angiogram in our institute. There were 110 angiographically proven patients of coronary artery disease with mean age of 55.8 +/- 9 years (range 34-72 years) and 75 subjects with normal coronary artery anatomy with mean age of 54.8 +/- 8 years (range 34-68 years). The mean carotid intimal medial thickness of subjects with coronary artery disease was significantly higher than in subjects without coronary artery disease (0.84 +/- 0.16 mm v. 0.65 +/- 0.15 mm, p<0.001). The mean carotid intimal medial thicknesses in patients with triple vessel, double vessel and single vessel disease were 0.96 +/- 0.12 mm, 0.84 +/- 0.11 mm and 0.78 +/- 0.13 mm, respectively (p=0.05). The mean lipoprotein(a) of subjects with coronary artery disease was significantly higher than in subjects without coronary artery disease (35.9 +/- 22.3 mg/dl v. 19.1 +/- 21.2 mg/dl, p<0.001). Mean lipoprotein(a) levels in subjects with carotid intimal medial thickness <0.80 was 26.4 +/- 24.2 mg/dl and in subjects with carotid intimal medial thickness > or = 0.80 was 32.1 +/- 22.1 mg/dl (p=0.05). CONCLUSIONS: There is a strong correlation between carotid and coronary atherosclerosis and carotid intimal medial thickness is a good predictor of presence and extent of coronary artery disease. Lipoprotein(a) level is a powerful independent risk factor for atherosclerosis. Carotid intimal medial thickness and lipoprotein(a) in conjoint can predict coronary artery disease reliably.

Plasma lipoprotein(a) levels in patients with pulmonary arterial hypertension.

BACKGROUND: Pulmonary artery hypertension is a common sequelae of a variety of cardiac and lung diseases. Pathogenesis of primary and secondary pulmonary artery hypertension is still debatable. METHODS AND RESULTS: We studied the serum lipoprotein(a) levels in patients with primary (n=27) and secondary (n=19) pulmonary artery hypertension (Eisenmenger syndrome). The results were compared with age and sex matched controls (n=46). We also studied the frequency of high levels of lipoprotein(a) (> 30 mg/dl) in pulmonary artery hypertension. Mean lipoprotein(a) levels were significantly higher in the pulmonary artery hypertension group compared to age- and sex-matched controls (31.60+/-15.49 mg/dl v. 14.66+/-14.7; p=0.0001). All patients were classified into two groups on the basis of their lipoprotein(a) levels (<30 mg/dl and >30 mg/dl). There was a higher frequency of lipoprotein(a) >30 mg/dl in patients of pulmonary artery hypertension v. controls (52% v. 24%; p= <0.001). Younger age, higher functional class, more severe congestive heart failure, shorter duration of symptoms. and more cases of hemoptysis were observed in the group with lipoprotein(a) >30 mg/dl. CONCLUSIONS: High lipoprotein(a) may be a marker and be associated with a more adverse prognosis in severe pulmonary artery hypertension. Larger prospective studies are needed to establish lipoprotein(a) as a risk factor for the development of pulmonary artery hypertension.

Short-term androgen deprivation does not alter CaR and VDR mRNA expression in duodenal mucosa in male rats.

Androgen deprivation is associated with decline in intestinal calcium absorption. The effect of androgen on CaR and VDR intestinal mucosa has not yet been studied. Calcium homeostasis, a real bone mineral density (aBMD, dual energy X-ray absorptiometry) and expression of CaR and VDR mRNA in duodenal mucosa of orchidectomized (ORX) and sham operated (Sham) adult Sprague Dawley rats at 4 week have been studied. There was no significant difference in serum calcium, alkaline phosphatase, calcidiol and calcitriol levels between both the groups. Serum testosterone (T) (ng/dl) and inorganic phosphorous (iP) (mg/dl) levels were significantly lower in ORX rats. As compared to sham rats, ORX rats had significant decline in in-vitro aBMD at proximal, middle and distal tibia, proximal, mid and distal femur and femoral neck (P < 0.05). Northern blot analysis revealed no significant alteration in the CaR and VDR mRNA expression in duodenal mucosa in ORX rats. CaR and VDR mRNA expression in duodenal mucosa is therefore, not affected by physiological concentrations of testosterone in rats.

Effect of oophorectomy on expression of calcium sensing receptor mRNA in rat duodenal mucosa.

Calcium sensing receptor (CaR) in duodenal mucosa may be involved in active calcium absorption. Estrogen deficiency results in decreased intestinal calcium absorption. Effects of bilateral oophorectomy (OVX) have been studied on calcium homeostasis, bone mineral density (BMD) and CaR mRNA levels in duodenal mucosa at 4 weeks in adult female Sprague Dawley rats and compared with those in sham-operated and control group. There was no significant change in serum corrected calcium, inorganic phosphorous, calcidiol and intact parathyroid hormone in all the three groups. OVX rats had a significant decline in serum estrogen (E2) levels and alkaline phosphatase. They also had a significant decrease in BMD (DXA) at lumbar spine in vivo, and proximal and distal tibia in vitro while there was no significant change in serum E2 and BMD parameters in sham-operated and control rats. Northern blot analysis revealed no significant change in the CaR mRNA expression in duodenal mucosa in all three groups. The results suggests that CaR mRNA expression in duodenal mucosa is not affected by physiological circulating concentrations of estradiol in rats.

Vitamin D and insulin resistance in postmenopausal Indian women.

PURPOSE: The purpose of this study is to investigate the association of the serum 25-hydroxyvitamin D (25-OHD) level with markers of insulin resistance (IR) in postmenopausal Indian women. MATERIALS AND METHODS: This was a cross-sectional study, conducted at a Tertiary Care Hospital in New Delhi, India. Seventy one postmenopausal women (mean age 56.3 ± 7.6 years) were enrolled. Exclusion criteria were known or newly detected diabetics, subjects with chronic renal failure, chronic liver disease or any other chronic inflammatory condition, chronic smokers and chronic alcoholics. Serum calcium (and albumin for calculating corrected calcium), phosphorus, alkaline phosphatase and 25-OHD were measured as parameters of calcium homeostasis. Fasting blood glucose (FBG), systolic and diastolic blood pressures, body mass index (BMI), fasting serum insulin, calculated glucose insulin ratio (GIR), and homeostatic model assessment of insulin resistance (HOMA-IR) were studied as parameters of IR. Data was then analyzed for statistical significance. RESULTS: The mean serum 25-OHD level was 12.73 ± 7.63 ng/ml. The mean BMI was 27.78 ± 5.37 kg/m(2). The mean calculated GIR was 13.14 ± 9.39 and HOMA-IR was 2.31 ± 1.70. Serum 25-OHD was inversely correlated with BMI (correlation coefficient -0.234, P value 0.050) and with HOMA-IR (correlation coefficient -0.237, P value 0.047). However, when 25-OHD was adjusted for BMI the correlation between 25-OHD and HOMA-IR lost its significance. No correlation was found between serum 25-OHD and any other parameters of IR studied. CONCLUSIONS: There is a significant negative linear correlation between 25-OHD and BMI. The significant negative linear correlation between 25-OHD and HOMA-IR was confounded by BMI. There is no correlation between 25-OHD and parameters of IR.

QSAR guided semi-synthesis and in-vitro validation of anticancer activity in ursolic acid derivatives.

As a part of our anticancer drug discovery programme, QSAR models were developed for the prediction of anticancer activities of ursolic acid derivatives against the human hepatocellular carcinoma HepG2, breast carcinoma MDA-MB-231 and the human ductal breast epithelial T47D cancer cell lines followed by wet lab semi-synthesis of virtually active derivatives, their in-vitro biological evaluation and apoptosis. The development of QSAR models was carried out by forward stepwise multiple linear regression method using a leave-one-out approach. Virtually active derivatives were semi synthesized, spectroscopically characterized and then in-vitro tested against human cancer cell lines. Active derivatives were checked via DNA fragmentation assay. The results exhibited regression coefficients (r(2)) and the cross-validation regression coefficients (rCV(2)) for the human HepG2, MDA-MB-231 and T47D cancer cell lines as .95 and .90; .92 and .87; .89 and .83 respectively showing the prediction accuracy of the models against biological activities. Computational molecular modeling is a valid modern approach, widely used in the identification of potential drug leads. The most active virtual derivatives of UA were semi- synthesized and their in-vitro and ex-vivo evaluation showed similar results with the predicted one, validating our QSAR models. Out of several active derivatives, the three UA2, UA7 and UA10 were potentially active against the above human cancer cell lines. These findings may be of immense importance in the anticancer drug development of an inexpensive and widely available natural product, ursolic acid.