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1.  Formation of iodoprotein during the peripheral metabolism of 35,3′-triiodo-l-thyronine-125I in the euthyroid man and rat 
Journal of Clinical Investigation  1969;48(4):685-695.
3,5,3′-Triiodo-L-thyronine-125I (T3-125I) metabolism was studied in nine euthyroid human subjects on blocking doses of nonradioactive iodide. After the intravenous injection of T3-125I, the fractional disappearance rate of plasma radioactivity progressively disappearance rate of plasma radioactivity progressively decreased with time. Analysis of individual plasma samples by dialysis, electrophoretic, and extraction techniques revealed three radioactive components: T3-125I, iodide-125I, and an unidentified material which was nonextractable in acid butanol (NE125I). Ne125I rose to maximal levels 24-36 hr after injection of T3-125I and then decreased with a fractional rate which approached, after 12-14 days, approximately 0.05 day-1 (t½ = 14 days). The plasma T3-125I concentration, obtained by subtraction of iodide-125I and NE125I from the plasma total 125I, declined at a constant fractional rate with time with a t½ of 1.5 days. Qualitatively similar results were obtained in rats. After 72 hr, 57% of the plasma and 40% of the liver radioactivity was NE125I. Chromatographic purification of the T3-125I before injection did not alter these results. The extrathyroidal origin of NE125I was further demonstrated by similar results in thyroidectomized rats maintained on thyroxine. NE125I from human sera separated from the other radioiodinated substances by ion-exchange chromatography was quantitatively precipitated by trichloracetic acid, not dialyzable, insoluble in CHCl3:CH2OH, and migrated with albumin during starch-gel electrophoresis. Based on these properties, NE125I was tentatively identified as iodoalbumin. Observations in rats equilibrated with 125I, as well as nonradioactive iodine determinations in human sera before and after acid butanol extraction, indicate that 10-20% of the serum organic iodine is in the form of iodoprotein. Our studies suggest that this moiety may be derived at least in part from the peripheral metabolism of the thyroid hormones.
PMCID: PMC322273  PMID: 5774106
2.  The preventive effect of N-butanol fraction of Nigella sativa on ethylene glycol-induced kidney calculi in rats 
Pharmacognosy Magazine  2011;7(28):338-343.
The current study was carried out to determine whether the aqueous-ethanolic extract or the butanolic fraction of Nigella sativa (NS) seeds could prevent or reduce calculi aggregation in experimental calcium oxalate nephrolithiasis in Wistar rats.
Materials and Methods:
Male Wistar rats were randomly divided into 5 groups: group A received tap drinking water for 28 days. Groups B, C, D and E received 1% ethylene glycol for induction of calcium oxalate (CaOx) calculus formation for 28 days. Rats in groups C, D and E also received aqueous-ethanolic extract of NS, N-butanol fraction and N-butanol phase remnant of NS, respectively, in drinking water at a dose of 250 mg/kg for 28 days. Urine concentration of oxalate, citrate, and calcium on days 0, 14, and 28, and also serum concentration of magnesium and calcium on days 0 and 28, were measured. On day 29, kidneys were removed for histopathologic study and examined for counting the calcium oxalate deposits in 10 microscopic fields.
Treatment of rats with N-butanol fraction and N-butanol phase remnant of NS significantly reduced the number and size of kidney calcium oxalate deposits compared with ethylene glycol group. Urinary concentration of oxalate in all experimental groups increased compared with control group on days 14 and 28, whereas the urine citrate concentration was lower in all experimental groups compared with control group on days 14 and 28.
N-butanol fraction and N-butanol phase remnant of NS showed a beneficial effect on calcium oxalate deposition in the rat kidney. Therefore, the butanolic fraction of NS may be suggested for prevention of calcium oxalate calculi in humans.
PMCID: PMC3261069  PMID: 22262938
Calcium oxalate; ethylene glycol; kidney calculi; N-butanol fraction; Nigella sativa
3.  Tomato fruits: a good target for iodine biofortification 
Iodine is a trace element that is fundamental for human health: its deficiency affects about two billion people worldwide. Fruits and vegetables are usually poor sources of iodine; however, plants can accumulate iodine if it is either present or exogenously administered to the soil. The biofortification of crops with iodine has therefore been proposed as a strategy for improving human nutrition. A greenhouse pot experiment was carried out to evaluate the possibility of biofortifying tomato fruits with iodine. Increasing concentrations of iodine supplied as KI or KIO3 were administered to plants as root treatments and the iodine accumulation in fruits was measured. The influences of the soil organic matter content or the nitrate level in the nutritive solution were analyzed. Finally, yield and qualitative properties of the biofortified tomatoes were considered, as well as the possible influence of fruit storage and processing on the iodine content. Results showed that the use of both the iodized salts induced a significant increase in the fruit’s iodine content in doses that did not affect plant growth and development. The final levels ranged from a few mg up to 10 mg iodine kg - 1 fruit fresh weight and are more than adequate for a biofortification program, since 150 μg iodine per day is the recommended dietary allowance for adults. In general, the iodine treatments scarcely affected fruit appearance and quality, even with the highest concentrations applied. In contrast, the use of KI in plants fertilized with low doses of nitrate induced moderate phytotoxicity symptoms. Organic matter-rich soils improved the plant’s health and production, with only mild reductions in iodine stored in the fruits. Finally, a short period of storage at room temperature or a 30-min boiling treatment did not reduce the iodine content in the fruits, if the peel was maintained. All these results suggest that tomato is a particularly suitable crop for iodine biofortification programs.
PMCID: PMC3694224  PMID: 23818889
biofortification; iodine; iodine deficiency; potassium iodate; potassium iodide; Solanum lycopersicum L.; tomato
4.  Antithyroid effects of lithium 
Journal of Clinical Investigation  1970;49(7):1357-1367.
Lithium has been reported to be goitrogenic when used for the treatment of manic-depressive psychosis. To investigate the effects of lithium on iodine metabolism, male Sprague-Dawley rats were placed on a low iodine (LID) or normal iodine diet (NID) containing enough Li2CO3 to give serum lithium levels of 0.23-0.86 mEq/liter (human therapeutic range is 0.6-1.6 mEq/liter). The following effects were noted with lithium treatment: (a) thyroid weight increased concomitant with a slowing of thyroidal iodine release; (b) the ability to concentrate iodide was increased only after goiters were established; (c) on the LID, 131I uptake was elevated throughout all phases of treatment, even when the release rate was normal; (d) iodine organification was unaffected but the proportion of 131I present as iodothyronines was decreased; (e) the thyroidal 127I content was increased; (f) despite these changes, the serum PBI remained normal as did the thyroxine turnover rate; and (g) thyrotropin (TSH) levels in serum were the same as controls except for a slight elevation early in the course of treatment; TSH levels did not correlate with goitrogenesis.
When LiCl was injected in large doses into intact rats (giving serum lithium levels of 3.08-3.89 mEq/liter), the iodide concentrating mechanism, 131I uptake, and 131I release rates were depressed. Similar experiments in hypophysectomized rats receiving TSH demonstrated these to be local antithyroid effects not mediated through the pituitary.
The discrepancy between acute and chronic responses to lithium, and the dissociation between the inhibition of iodine release and stimulatory effects is discussed.
PMCID: PMC322608  PMID: 4194189
5.  Antioxidant activities of saponins extracted from Radix Trichosanthis: an in vivo and in vitro evaluation 
Radix Trichosanthis (RT), the dry root tuber of Trichosanthis kirilowii Maxim (Cucurbitaceae), is a traditional Chinese medicine. Although a wide range of saponin pharmacological properties has been identified, to our knowledge, this may be the first report to investigate the crude saponins from RT. The purpose of this study was to delineate the antioxidant activity both in vitro and in vivo by using ethyl acetate (EtOAc), n-butanol, and the mixture of n-butanol and EtOAc fractions.
In vitro antioxidant activity was detected by using DPPH free radical, hydrogen peroxide scavenging, and reducing power assays. After pretreatment with different fractions saponins at 2 mg/kg/d and 3 mg/kg/d of crude drug, respectively, an established CCl4 induced acute cytotoxicity model was used to evaluate the in vivo antioxidant potential by detection of superoxide dismutase (SOD), malonaldehyde (MDA), lactate dehydrogenase (LDH), and total antioxidant capacity (T-AOC) levels.
The in vitro assay showed that the antioxidant activity of all the three fractions was promising. The reducing power of the EtOAc and the mixture of n-butanol and EtOAc extracts increased in a dose dependent manner. However, both the n-butanol and the mixture of n-butanol and EtOAc fractions in low dose exhibited in a time dependent manner with prolonged reaction time. As for hydrogen peroxide scavenging capability, the n-butanol fraction mainly demonstrated a time dependent manner, whereas EtOAc fraction showed a dose dependent manner. However, in case of in vivo assay, an increase of SOD and T-AOC and decrease of MDA and LDH levels were only observed in n-butanol (2 mg/kg/d of crude drug) extracts pretreatment group.
RT saponins in n-butanol fraction might be a potential antioxidant candidate, as CCl4-induced oxidative stress has been found to be alleviated, which may be associated with the time dependent manner of n-butanol saponins in a low dose. Further studies will be needed to investigate the active individual components in n-butanol extract, in vivo antioxidant activities and antioxidant mechanisms.
PMCID: PMC3973866  PMID: 24597831
Antioxidant; Liver; Radix trichosanthis; Radical scavenger; Saponins
6.  A radioimmunoassay for serum rat thyroglobulin. Physiologic and pharmacological studies. 
Journal of Clinical Investigation  1975;56(5):1073-1081.
A double antibody radioimmunoassay has been developed to measure thyroglobulin in rat (RTg) serum. The lowest detectable quantity measurable was 5.0 ng/ml. Specificity was documented by: (a) fall in serum RTg to undetectable levels after thyroid ablation; (b) the fact that L-thyroxine, D-thyroxine, L-triiodothyronine, D-triiodothyronine, triiodothyroacetic acid, tetraiodothyroacetic acid, triiodothyropropionic acid, moniodotyrosine, diiodotyrosine, and human thyroglobulin (HTg) in concentrations up to 40,000 ng per tube did not cross-react in the assay; (c) the demonstration that constant levels of serum RTg were observed while varying amounts of serum (criterion of parallelism) were introduced in the assay. The mean RTg concentration in tail vein blood of adult Sprague-Dawley rats were 101.5 +/- 13.0 ng/ml (SEM) (n=21); values ranged from 12.0 to 258.0 ng/ml. Chronic administration of a high-iodine diet (HID) did not affect serum thyroglobulin levels. Chronic administration of a low-iodine diet (LID) and propylthiouracil (PTU) led to a statistically significant increase in serum RTg that was accompanied by a significant rise in serum thyrotropin (rTSH). Serum thyroxine (T4) administered to normal rats for 14 days (20 mug/day subcutaneously) depressed serum RTg concentration from a mean level of 119.4 +/- 17.5 ng/ml (n=19) to a mean of 35.0 +/- 0.27 ng/ml (n=19) (P less than 0.001). While rats were on continuous T4 suppression, bovine thyroid-stimulating hormone (bTSH) given intravenously (2 IU) resulted in a mean maximal increment of RTg of 332.0 +/- 81.5 ng/ml (n=6) at 24 h. IgC-(LATS) long-acting thyroid stimulatory injected intravenously resulted in a mean maximal increment of RTg concentration at 96 h of 87.2 +/- 14.3 ng/ml (n=5). Normal IgG had no statistical significant effect of RTg levels at any time after the injection.
PMCID: PMC301968  PMID: 1184735
7.  Detection of Schistosoma mansoni Membrane Antigens by Immunoblot Analysis of Sera of Patients from Low-Transmission Areas 
Schistosoma mansoni surface membrane components play a relevant role in the host-parasite interaction, and some are released in vivo as circulating antigens. n-Butanol extraction favors the release of membrane antigens like alkaline phosphatase, which has been shown to be specifically recognized by antibodies from S. mansoni-infected humans and animals. In the present study, components in the n-butanol extract (BE) of the adult S. mansoni worm membrane fraction were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D SDS-PAGE [15%]) and further analyzed by immunoblotting (immunoglobulin G) using defined sera. S. mansoni-infected patient sera, but not sera of uninfected patients or sera obtained from patients infected with other parasite species, specifically and variably recognized up to 20 polypeptides in the molecular mass range of ∼8 to >80 kDa. There were some differences in the number, intensity, and frequency of recognition of the BE antigens among sera from Venezuelan sites of endemicity with a different status of schistosomiasis transmission. Antigens in the 28- to 24-kDa molecular mass range appeared as immunodominants and were recognized by S. mansoni-positive sera from all the sites, with recognition frequencies varying between 57.5 and 97.5%. Immunoblotting with BE membrane antigens resulted in a highly sensitive (98.1%), specific (96.1.0%), and confirmatory test for the immunodiagnosis of schistosomiasis in low-transmission areas.
PMCID: PMC549304  PMID: 15699423
8.  Usefulness of low iodine diet in managing patients with differentiated thyroid cancer - initial results 
Radiology and Oncology  2011;45(3):189-195.
Low iodine diet (LID) is recommended in patients with differentiated thyroid cancer before radioiodine administration. Patients with increased thyroglobulin (Tg) level, but negative 131I whole body scan present diagnostic and therapeutic dilemma. This study was designed to evaluate the benefit of a two-week LID in patients with elevated serum Tg levels and negative 131I whole body scans.
Patients and methods.
For the impact assessment of two-week LID on radioiodine tissue avidity, radioiodine scans before and after LID were compared. Sixteen patients with serum Tg > 2 μg/L, negative Tg-antibodies, and negative radioiodine scans underwent two-week LID before the 131I administration. Fourteen patients underwent diagnostic scanning and two patients received radioiodine therapy. Iodine concentration in the morning urine specimens were measured in each patient, a day before and 15th day after starting LID.
Following self-managed LID, patients were able to significantly reduce their iodine body content by 50% (range 28–65%, p<0,001). 13 patients (82%) accomplished mild iodine deficiency (50-99 μg/L) and one patient (6%) achieved targeted moderate iodine deficient state (<50 μg/L). All diagnostic post-LID scans were negative. Both post-therapy 131I scans showed radioiodine accumulation outside of normal 131I distribution (neck region and diffuse hepatic uptake). This study demonstrated that two-week LID is effective way to decrease total body iodine content, although without a visible effect on post-LID diagnostic 131I scans.
A more stringent dietary protocol and longer iodine restriction period are probably needed to achieve targeted moderate iodine deficiency in patients preparing for 131I administration. This might result in higher radioiodine avidity of thyroid remnant/metastases.
PMCID: PMC3423737  PMID: 22933955
low iodine diet; urine iodine concentration; differentiated thyroid cancer; radioiodine
9.  Iodine deficiency, hypothyroidism, and endemic goitre in southern Tanzania. A survey showing the positive effects of iodised oil injections by TSH determination in dried blood spots. 
The Ukinga and Uwanji regions, located in the southern highlands of Tanzania, were studied for the degree of iodine deficiency and the incidence of goitre and hypothyroidism, respectively. A urinary iodine excretion as low as 17.6 +/- 9.3 micrograms/g creatinine was observed in Wangama village. The mean goitre prevalence in 27 villages in Uwanji ranged between 65 and 96% (n = 3031 schoolchildren). Of 681 pregnant women from Ukinga 79.6% had goitre. The prevalence of cretinism as estimated on clinical criteria was 3% in Magoye (Uwanji). A normal serum TSH (below 2.1 mU/l) was observed in only 12 out of 66 school children before iodine prophylaxis, whereas the T4/TBG ratio was decreased in 36 of 63 cases. Blood spot TSH levels in newborn infants (n = 219) from mothers without iodine supplementation were above 12 mU/l in 45%. In contrast, only 20.3% of the newborn (n = 118) had elevated blood spot TSH (p less than 0.002) when the mothers had received an iodised oil injection during pregnancy. Most of the newborn (n = 18; 75%) of the latter group with elevated TSH (n = 24) came from mothers who had received the iodine injection only 1-25 days before delivery. Maternal iodine prophylaxis in late pregnancy does not increase the rate of neonatal hypothyroidism. Conclusions: It has been confirmed that severe iodine deficiency resulting in endemic goitre, cretinism, and hypothyroidism is prevalent in the regions studied. Dried blood spot TSH determinations may serve as an index for the efficiency of iodine prophylaxis programmes. Such a programme was carried out with relatively little expenditure and effort on a large scale basis.
PMCID: PMC1052447  PMID: 2995534
10.  Materials Indistinguishable from Iodotyrosines in Normal Human Serum and Human Serum Albumin* 
Journal of Clinical Investigation  1967;46(7):1264-1274.
Materials indistinguishable from authentic mono- and diiodotyrosines were identified in extracts of normal human serum as well as in extracts of purified human serum albumin. These materials were not found in association with the other serum proteins. Identification of MIT and DIT was made by a technique using rechromatography to constant specific activity, as well as by the Barker wet ash distillation method, which established the compounds in question as being iodinated ones. By two different extraction and chromatographic methods we estimated the amounts of both MIT and DIT present in normal human serum or albumin; the estimates were in good agreement. These compounds together constituted between 19% and 25% of the extractable serum iodine.
PMCID: PMC297125  PMID: 6027088
11.  Identification of a potent serum factor that causes desensitization of the receptor for C-Type natriuretic peptide 
Guanylyl cyclase-B (GC-B; NPR-B), the receptor for C-type natriuretic peptide (CNP) is rapidly and effectively desensitized by a factor(s) in serum. Given the potential importance of this receptor in remodeling after tissue injury, identification of the serum factor(s) is of significant medical importance.
Partial purification of desensitization activity in serum by DEAE-Sepharose and reverse phase C18 chromatography, followed by mass spectroscopy, identified peptide sequences identical to those of apolipoprotein A2 (Apo A2), a known component of high density lipoprotein (HDL). Apo A2, however, could be eliminated as the active desensitization factor. Never the less, substantial desensitization activity was associated with purified preparations of bovine or human HDL. Since HDL is a well-known transporter of various lipids and phospholipids, we extracted either HDL or partially purified serum preparations with butanol and all activity extracted into the solvent. Of various lipophilic signaling molecules known to be associated with HDL, a prominent component is sphingosine-1-phosphate (S1P). We therefore tested authentic S1P as well as other known components of HDL (sphingosylphosphorylcholine; platelet activating factor) for activity; only S1P caused desensitization of GC-B. S1P was relatively potent, causing one-half maximal desensitization of GC-B at concentrations of 5–10 nM. These effects were seen within a few minutes after addition. Lysophosphatidic acid, another component of serum capable of desensitizing GC-B, was only effective at Micromolar concentrations. The pathway by which serum or S1P desensitizes GC-B seems unique in that pertussis toxin failed to inhibit GC-B desensitization, and yet blocked serum or S1P activation of extracellular signal-regulated kinase (ERK) or Akt/protein kinase B (Akt/PKB).
Since the concentrations of S1P that desensitize GC-B are well within serum physiological ranges, this mitogenic signaling molecule likely functions as a strong adversary of the CNP/GC-B signaling pathway in the regulation of cell proliferation and other growth factor-induced phenotypes. The mechanism by which S1P desensitizes GC-B appears different than the known S1P signaling pathways.
PMCID: PMC305373  PMID: 14627441
12.  Influence of dietary iodine deficiency on the thyroid gland in Slc26a4-null mutant mice 
Thyroid Research  2011;4:10.
Pendred syndrome (PDS) is an autosomal recessive disorder characterized by sensorineural hearing impairment and variable degree of goitrous enlargement of the thyroid gland with a partial defect in iodine organification. The thyroid function phenotype can range from normal function to overt hypothyroidism. It is caused by loss-of-function mutations in the SLC26A4 (PDS) gene. The severity of the goiter has been postulated to depend on the amount of dietary iodine intake. However, direct evidence has not been shown to support this hypothesis. Because Slc26a4-null mice have deafness but do not develop goiter, we fed the mutant mice a control diet or an iodine-deficient diet to evaluate whether iodine deficiency is a causative environmental factor for goiter development in PDS.
We evaluated the thyroid volume in histological sections with the use of three-dimensional reconstitution software, we measured serum levels of total tri-iodothyronine (TT3) and total thyroxine (TT4) levels, and we studied the thyroid gland morphology by transmission electron microscopy.
TT4 levels became low but TT3 levels did not change significantly after eight weeks of an iodine-deficient diet compared to levels in the control diet animals. Even in Slc26a4-null mice fed an iodine-deficient diet, the volume of the thyroid gland did not increase although the size of each epithelial cell increased with a concomitant decrease of thyroid colloidal area.
An iodine-deficient diet did not induce goiter in Slc26a4-null mice, suggesting that other environmental, epigenetic or genetic factors are involved in goiter development in PDS.
PMCID: PMC3141755  PMID: 21689387
13.  Selumetinib-Enhanced Radioiodine Uptake in Advanced Thyroid Cancer 
The New England journal of medicine  2013;368(7):623-632.
Metastatic thyroid cancers that are refractory to radioiodine (iodine-131) are associated with a poor prognosis. In mouse models of thyroid cancer, selective mitogen-activated protein kinase (MAPK) pathway antagonists increase the expression of the sodium–iodide symporter and uptake of iodine. Their effects in humans are not known.
We conducted a study to determine whether the MAPK kinase (MEK) 1 and MEK2 inhibitor selumetinib (AZD6244, ARRY-142886) could reverse refractoriness to radioiodine in patients with metastatic thyroid cancer. After stimulation with thyrotropin alfa, dosimetry with iodine-124 positron-emission tomography (PET) was performed before and 4 weeks after treatment with selumetinib (75 mg twice daily). If the second iodine-124 PET study indicated that a dose of iodine-131 of 2000 cGy or more could be delivered to the metastatic lesion or lesions, therapeutic radioiodine was administered while the patient was receiving selumetinib.
Of 24 patients screened for the study, 20 could be evaluated. The median age was 61 years (range, 44 to 77), and 11 patients were men. Nine patients had tumors with BRAF mutations, and 5 patients had tumors with mutations of NRAS. Selumetinib increased the uptake of iodine-124 in 12 of the 20 patients (4 of 9 patients with BRAF mutations and 5 of 5 patients with NRAS mutations). Eight of these 12 patients reached the dosimetry threshold for radioiodine therapy, including all 5 patients with NRAS mutations. Of the 8 patients treated with radioiodine, 5 had confirmed partial responses and 3 had stable disease; all patients had decreases in serum thyroglobulin levels (mean reduction, 89%). No toxic effects of grade 3 or higher attributable by the investigators to selumetinib were observed. One patient received a diagnosis of myelodysplastic syndrome more than 51 weeks after radioiodine treatment, with progression to acute leukemia.
Selumetinib produces clinically meaningful increases in iodine uptake and retention in a subgroup of patients with thyroid cancer that is refractory to radioiodine; the effectiveness may be greater in patients with RAS-mutant disease. (Funded by the American Thyroid Association and others; number, NCT00970359.)
PMCID: PMC3615415  PMID: 23406027
The numbers of adults with diabetes in the world is estimated to rise 300 millions in the year 2025 and this leads to increasing search for better anti-diabetic drug. The effects of the ethanol extract (1.25 g/kg) and fractions (Ethyl acetate, dichloromethane and butanol in the dose of 1 g/kg) of Anacardium occidentale stem bark on the blood glucose levels in streptozotocin-induced types 1 and 2 diabetic rats at different prandial states were studied. The ethanol extract of A. occidentale had no hypoglycemic effect in type 1 diabetic rats in fasting and postprandial glucose load conditions and, in type 2 diabetic rats in fasting condition. However, the extract, significantly lowered blood glucose levels in type 2 diabetic rats when fed simultaneously with glucose. The ethyl acetate fraction showed a significant opposing effect in serum glucose rise after administration of glucose. Additionally, its dichloromethane extract also exhibited a significant reduction in serum glucose level compared to control after glucose administration while its butanol fraction was devoid of this activity. These findings conclude that the active principles responsible for the antihyperglycaemic effect might be concentrated in the ethyl acetate and dichloromethane fractions of the extract.
PMCID: PMC4158895  PMID: 25206248
Anacardium occidentale; Non insulin dependent diabetes; streptozotocin
15.  A multiple ligand-binding radioimmunoassay of diiodotyrosine. 
Journal of Clinical Investigation  1974;53(2):416-422.
A radioimmunoassay has been developed for the measurement of 3,5-diiodo-L-tyrosine (DIT) in serum. DIT was coupled to porcine thyroglobulin (PTg) with a molar ratio of 205:1. Rabbits were immunized with 1 mg of immunogen emulsified in complete Freund's adjuvant. Sera were screened for their ability to bind trace amounts of [125I]DIT. A serum that bound 40% of the tracer at a final dilution of 1:1,750 was used in the assay. Assay specificity was improved by the use of thyroxine (T4)-binding globulin as a second ligand-binding protein to decrease T4 and triiodothyronine (T3) cross-reactivity with the antibody. Double antibody and polyethylene glycol radioimmunoassays were compared. DIT present in the second antiserum shifted the double antibody assay standard curve and altered estimates of assay specificity and assay sensitivity. By using the polyethylene glycol system and butanol:ethanol extracts of serum, DIT was measured in human serum. In 35 apparently healthy young adult controls DIT levels averaged 156 ng/100 ml. Random DIT levels averaged 158 ng/100 ml in 11 untreated hyperthyroid patients and 84 ng/100 ml in 15 untreated primary hypothyroid patients. No diurnal pattern in DIT levels could be demonstrated. Thyroid-stimulating hormone administration led to a variable but small rise in DIT levels, but short term T3 suppression was not associated with a measurable fall in DIT concentrations. Paired serum samples from the carotid artery and thyroid vein of 10 euthyroid goiter patients and one patient with a toxic solitary adenoma all showed a positive transthyroidal gradient indicating the thyroidal release of DIT in each patient. Measurable DIT levels of 45, 47, 68, and 80 ng/100 ml, respectively, were found in four fasting athyrotic patients indicating that the thyroid is not the only source of serum DIT.
PMCID: PMC301484  PMID: 11344555
16.  The Prevalence of Thyroid Dysfunction in Elderly Cardiology Patients with Mild Excessive Iodine Intake in the Urban Area of São Paulo 
Clinics (Sao Paulo, Brazil)  2009;64(2):135-142.
To evaluate the prevalence of thyroid dysfunction in elderly cardiac patients in an outpatient setting.
A total of 399 consecutive patients (268 women, age range 60–92 years) who were followed at Heart Institute were evaluated for thyroid dysfunction with serum free T4, TSH, anti-Peroxidase antibodies, urinary iodine excretion measurements and thyroid ultrasound.
Hyperthyroidism (overt and subclinical) was present in 29 patients (6.5%), whereas hypothyroidism (overt and subclinical) was found in 32 individuals (8.1%). Cysts were detected in 11 patients (2.8%), single nodules were detected in 102 (25.6%), and multinodular goiters were detected in 34 (8.5%). Hashimoto’s thyroiditis was present in 16.8% patients, most of whom were women (83.6%). The serum TSH increased with age and was significantly higher (p= <0.01) in patients, compared to the normal control group. No significant differences in serum TSH and free T4 values were observed when patients with atrial fibrillation (AF) where compared with those without arrhythmia. The median urinary iodine levels were 210 μg/L (40–856 μg/L), and iodine levels were higher in men than in women (p<0.01). Excessive iodine intake (urinary iodine >300 μg/L) was observed in one-third of patients (30.8%).
Elderly patients have a higher prevalence of both hypo- and hyperthyroidism as well as thyroid nodules when compared with the general population. About one-third of the older patients had elevated urinary secretion of iodine and a higher prevalence of chronic Hashimoto’s thyroiditis. It is recommended that ultrasonographic studies, tests for thyroid function and autoimmunity should be evaluated in elderly patients.
PMCID: PMC2666482  PMID: 19219319
Iodine intake; Thyroid dysfunction; Cardiologic patients; Elderly patients; Urinary iodine
17.  Butanol Fraction Containing Berberine or Related Compound From Nexrutine® Inhibits NFκB Signaling and Induces Apoptosis in Prostate Cancer Cells 
The Prostate  2009;69(5):494-504.
Epidemiological and laboratory studies support the hypothesis that several plant components influence prostate carcinogenesis and holds promise for disease prevention. Previously we reported that Nexrutine® (bark extract from Phellodendron amurense) inhibits proliferation of prostate cancer cells and prostate tumor development in the transgenic adenocarcinoma of mouse prostate (TRAMP) model through modulation of Akt signaling pathway. In the present investigation we conducted studies to further define the mechanism of action of Nexrutine® and to identify the active component associated with its biological activity.
Androgen-responsive, androgen-independent human prostate cancer cell lines and tissues from TRAMP mice fed Nexrutine® were used in these studies. Activity guided fractionation identified butanol fraction recapitulating the activities of Nexrutine® assessed by proliferation assays, apoptotic assays (DAPI and TUNEL staining), transient transfections, gel shift assays and Western blotting. In addition ultra-performance liquid chromatography (UPLC) of butanol fraction was used to identify active component of Nexrutine®.
Butanol fraction recapitulated the activities of Nexrutine® in (i) inhibiting proliferation; (ii) inducing apoptosis; and (iii) modulating transcriptional activity of NFκB in prostate cancer cells. Our data also indicates that both Nexrutine® and butanol fraction modulates NFκB transcriptional activity by inhibiting IκBα phosphorylation. Expression of p65 and phosphorylated IκBα are high in tumors from TRAMP mice. In contrast dietary administration of Nexrutine® reduced expression of p65 and phosphorylated IκBα in prostate from TRAMP mice. In addition using UPLC, we have identified berberine or closely related compound in the butanol fraction.
The results suggest that berberine or closely related component of butanol fraction may be responsible for the observed biological activities and induce apoptosis in prostate cancer cells by targeting critical cell survival signaling pathways both in vitro and in vivo.
PMCID: PMC2674392  PMID: 19107816
nexrutine; apoptosis; fractionation
18.  Anti-proliferative effects of Bifidobacterium adolescentis SPM0212 extract on human colon cancer cell lines 
BMC Cancer  2008;8:310.
Lactic acid bacteria (LAB) are beneficial probiotic organisms that contribute to improved nutrition, microbial balance, and immuno-enhancement of the intestinal tract, as well as anti-tumor activity. The aim of the present work was to study the growth inhibition of tumor cells by butanol extract of Bifidobacterium adolescentis isolated from healthy young Koreans.
The anti-proliferative activity of B. adolescentis isolates was assessed by XTT assays on three human colon cancer cell lines (Caco-2, HT-29, and SW480). The effects of B. adolescentis SPM0212 butanol extract on tumor necrosis factor-α (TNF-α) and nitric oxide (NO) production were tested using the murine macrophage RAW 264.7 cell line.
The butanol extract of B. adolescentis SPM0212 dose-dependently inhibited the growth of Caco-2, HT-29, and SW480 cells by 70%, 30%, and 40%, respectively, at 200 μg/mL. Additionally, the butanol extract of B. adolescentis SPM0212 induced macrophage activation and significantly increased the production of TNF-α and NO, which regulate immune modulation and are cytotoxic to tumor cells.
The butanol extract of B. adolescentis SPM0212 increased activity of the host immune system and may improve human health by helping to prevent colon cancer as a biological response modifier.
PMCID: PMC2605768  PMID: 18950540
19.  Influence of lipopolysaccharide on graft versus host reactivity of lipopolysaccharide-unresponsive C3H/HeJ mice. 
Infection and Immunity  1979;26(1):137-142.
It was initially reported that lipopolysaccharide (LPS)-unresponsive C3H/HeJ mice are refractory to LPS at the B-lymphocyte level, but more recently it has been shown that other cells are similarly unaffected. The current study was undertaken to study an in vivo LPS-modulated disease process involving macrophage-T cell interactions. Adult CBA/J and C3H/HeJ mice were used as spleen donors, and graft versus host reactions were induced in BALB/c neonates. Prior LPS treatment of CBA/J adults decreased the ability of their spleen cells to cause fatal graft versus host disease in BALB/c neonates, whereas no difference was found between injection of spleen cells from normal or LPS-treated C3H/HeJ mice. Similar results were obtained with these cell types when the mouse spleen mixed leukocyte culture system was used. In a carbon clearance assay for stimulation of the reticuloendothelial system with LPS, it was found that the rate of phagocytosis was significantly increased in BALB/c and CBA/J mice 72 h after inoculation of LPS. No stimulation was seen in rate of carbon uptake in the C3H/HeJ animals after treatment with phenol-extracted LPS or with butanol-extracted LPS. An LPS-induced protective serum factor was produced only in the LPS-responsive CBA/J mice and was specific for the syngeneic cells.
PMCID: PMC414585  PMID: 40877
20.  Alterations in thyroid hormone economy in patients with hydatidiform mole 
Journal of Clinical Investigation  1971;50(6):1345-1354.
Studies of several aspects of thyroid hormone economy have been conducted in 11 patients before and after removal of a molar pregnancy. Before evacuation of the mole, all patients demonstrated moderately to greatly elevated values for thyroidal 131I uptake, absolute iodine uptake, and serum protein-bound-131I. Values for serum PBI and serum thyroxine (T4) concentration were consistently and often greatly increased, averaging more than twice those found in normal pregnancy and three times those in normal controls. On the other hand, the maximum binding capacity of the T4-binding globulin (TBG) was variably affected, and ranged between the values found in normal controls and those found in normal pregnancy. Values for the absolute concentration of free T4 in serum were, on the average, only moderately elevated, since the proportion of free T4 was moderately low, although not as low as in normal pregnancy. Sera of patients with molar pregnancy contained high levels of thyroid stimulating activity, as assessed in the McKenzie mouse bioassay system. The stimulator displayed a more prolonged duration of action than that of TSH and did not reveal a major immunological cross-reactivity with either human or bovine TSH, differing in the latter respect from the chorionic thyrotropin of normal human placenta. Abnormalities in iodine metabolism were rapidly ameliorated after removal of the molar pregnancy, and this was associated with the disappearance from serum of the thyroid stimulator.
Despite the foregoing evidence of thyroid hyperfunction and hypersecretion of T4, patients with molar pregnancy were neither goitrous nor overtly thyrotoxic. They did display, however, high values of the urinary pigment/creatinine ratio, a possible indication of the presence of a hypermetabolic state.
It is concluded that in patients with molar pregnancy, thyroid function and T4 secretion are stimulated, often greatly so, by an unusual thyroid stimulator which is demonstrable in the blood and which is probably of molar origin. The nature of the stimulator, as well as the reasons for both the variability of the increase in TBG which occurs in molar pregnancy and the lack of frank thyrotoxicosis, remain to be clarified.
PMCID: PMC292066  PMID: 4102851
21.  Triiodothyronine and Thyroxine in the Serum and Thyroid Glands of Iodine-Deficient Rats 
Journal of Clinical Investigation  1973;52(10):2522-2531.
Triiodothyronine (T3) and thyroxine (T4) were measured by immunoassay in the serum and thyroid hydrolysates of control (group A), mildly iodine-deficient (group B), and severely iodine-deficient rats (group C). These results were correlated with changes in thyroidal weight, 131I uptake and 127I content as well as with the distribution of 131I in Pronase digests of the thyroid. There was a progressive increase in thyroid weight and 131I uptake at 24 h with decrease in iodine intake. The 127I content of the thyroids of the group B animals was 44% and that of the group C animals 2% of that in group A. The mean labeled monoiodotyrosine/diiodotyrosine (MIT/DIT) and T3/T4 ratios in group A were 0.42±0.07 (SD) and 0.12±0.01, 0.59±0.06 and 0.11±0.03 in group B, and 2.0±0.3 and 1.8±0.9 in the group thyroid digests.
Mean serum T4 concentration in the control rats was 4.2±0.6 (SD) μg T4/100 ml, 4.5±0.3 μg/100 ml in group B animals, and undectectable (<0.5 μ4/100 ml) in group C animals. There was no effect of iodine deficiency on serum T3 concentrations, which were 44±9 (Mean±SD) ng/100 ml in A animals, 48±6 ng/100 ml n B animals, and 43±6 ng/100 ml in the C group. Thyroidal digest T3 and T4 concentrations were 39 and 400 ng/mg in group A animals and were reduced to 5 and 1% of this, respectively, in group C. The molar ratio of T3/T4 in the thyroid digests of the groups A and B animals was identical to the ratio of labeled T3/T4 and was slightly less (1.0±0.9) than the labeled T3/T4 ratio in the group C animals.
The mean ratio of labeled T4 to labeled T3 in the serum of the severely iodine-deficient animals 24 h after isotope injection was 11±1 (SEM). With previously published values, it was possible to correlate the ratio of labeled T4/T3 in the thyroid digest with the labeled T4/T3 ratio in the serum of each iodine-deficient animal. This analysis suggested that the labeled thyroid hormones in the severely iodine-deficient rat were secreted in the ratio in which they are present in the gland.
Kinetic analysis of total iodothyronine turnover indicated that two-thirds of the T3 utilized per day by the iodine-sufficient rat arises from T4. If the T4-T3 conversion ratio remains the same in iodine deficiency, then the analysis suggests that about 90% of the T3 arises directly from the thyroid. Therefore, it would appear that absolute T3 secretion by the thyroid increases severalfold during iodine deficiency. The fact that serum T3 remains constant and T4 decreases to extremely low levels, combined with previous observations that iodine-deficient animals appear to be euthyroid, is compatible with the hypothesis that T4 in the normal rat serves primarily as a precursor of T3.
PMCID: PMC302511  PMID: 4729046
22.  A Radioimmunoassay for Measurement of 3,3′, 5′-Triiodothyronine (Reverse T3) 
Journal of Clinical Investigation  1974;54(3):583-592.
A highly specific antiserum to 3,3′,5′-triiodothyronine (reverse T3, rT3) was prepared by immunization of rabbits with D,L-rT3-human serum albumin conjugate. Of the various thyroid hormone derivatives tested, only 3,3′-diiodothyronine (3,3′-T2) cross-reacted significantly (10%) with rT3-binding sites on the antiserum, while thyroxine (T4) and triiodothyronine (T3) cross-reacted by less than 0.1%. The antiserum was used in a simple, sensitive, precise, and reproducible radioimmunoassay (RIA) for measurement of rT3 in ethanolic extracts of serum. The dose-response curves of inhibition of the binding of [125I]rT3 to antibody obtained by serial dilutions of serum extracts were essentially parallel to the standard assay curve. Recovery of nonradioactive rT3 added to serum before extraction averaged 93%.
Serum rT3 concentrations were found to be (mean±SD) 41±10 ng/100 ml in 27 normal subjects, 103±49 ng/100 ml in 22 hyperthyroid patients, 19±9 ng/100 ml in 12 hypothyroid patients, and 54±7 ng/100 ml in five subjects with elevated serum thyroxine-binding globulin: the values in each of the latter three groups of individuals were significantly different from normal. Reverse T3 was detected regularly in normal or supranormal concentrations in serum of 12 hypothyroid patients rendered euthyroid or mildly hyperthyroid by treatment with synthetic T4. It is suggested that serum rT3 values noted here should be taken to reflect the relative changes in serum rT3 rather than its absolute values in health and thyroid disease. True serum rT3 may be somewhat different because: (a) D.L-rT3 employed in the standard curve and L-rT3 present in human serum may react differently with anti-D,L-rT2. (b) Even though 3,3′-T2, which cross-reacted 10% in rT3 RIA, has been considered unlikely to be present in human serum, it may circulate in low levels. (c) Cross-reaction of T4 in rT3 RIA of 0.06% although small, could contribute to RIA estimates of rT2; the effect of T4 would be particularly important in case of serum of hyperthyroid patients. Thus, serum rT3 concentration in hyperthyroid patients averaged 89±48 μg/100 ml after correction for cross-reaction effects of T4: this value was about 14% lower than that before correction (see above).
Serum rT3 concentration in cord sera of seven newborns averaged 136±19 ng/100 ml; it was clearly elevated and within the range of values seen in hyperthyroid patients. This was the case when the mean T4 concentration in the newborn cord sera was moderately higher than normal and about one-half that in hyperthyroid patients, whereas serum T3 was markedly below the normal adult level. A Pronase hydrolysate of thyroglobulin prepared from pooled normal thyroid glands contained 0.042, 3.0, and 0.16 μg/mg protein of rT3, T4, and T3, respectively. The various data suggest that: (a) rT3 is a normal component of human serum and thyroglobulin: (b) peripheral metabolism of T4 is an important source of the rT3 present in serum: (c) peripheral conversion of T4 to T3 and rT3 may not necessarily be a random process.
PMCID: PMC301591  PMID: 4211761
23.  In Vitro Antibacterial Activity, Gas Chromatography–Mass Spectrometry Analysis of Woodfordia fruticosa Kurz. Leaf Extract and Host Toxicity Testing With In Vitro Cultured Lymphocytes From Human Umbilical Cord Blood 
To locate a plant with suitable phytochemicals for use as antimicrobial agents to control multidrug-resistant (MDR) bacteria as a complementary medicine, without host toxicity as monitored through cultured lymphocytes from human umbilical cord blood.
The methanol crude leaf extract of the plant Woodfordia fruticosa was subjected to antimicrobial assay in vitro with nine pathogenic MDR bacteria from clinical samples. This was followed by bioassay-guided fractionation with seven non-polar to polar solvents, gas chromatography–mass spectrometry analysis of the n-butanol fraction, and monitoring of the host toxicity of the leaf extract with in vitro grown lymphocytes from human umbilical cord blood.
The leaf extract of W. fruticosa had a controlling capacity for MDR bacteria. The minimum inhibitory concentration and minimum bactericidal concentration of the n-butanol fraction were < 1.89 mg/mL extract and 9.63 mg/mL extract, respectively. The gas chromatography–mass spectrometry spectrum of the n-butanol fraction confirmed the presence of 13 peaks of different compounds with retention times of 9.11 minutes, 9.72 minutes, 10.13 minutes, 10.78 minutes, 12.37 minutes, 12.93 minutes, 18.16 minutes, 21.74 minutes, 21.84 minutes, 5.96 minutes, 12.93 minutes, 24.70 minutes, and 25.76 minutes. The six leading compounds were: diethyl phthalate: IUPAC name: diethyl benzene-1,2-dicarboxylate; 5-methyl-2-(1-methylethyl) phenol: IUPAC name: 5-methyl-2-propan-2-ylphenol; (E )-3,7-dimethylocta-2,6-diene-1-thiol: IUPAC name: (2Z)-3,7-dimethylocta-2,6-diene-1-thiol; 2,6,10-dodecatrien-1-ol, 3,7,11-trimethyl-, (E,E ): IUPAC name: 2,6,10-dodecatrien-1-ol; 3,7,11-trimethyl-, (E,E); 2-methoxy-4-(2-propenyl) phenol: IUPAC name: 2-methoxy-4-[(1E)-prop-1-en-1-yl]phenol; hexadecanoic acid: IUPAC name: hexadecanoic acid.
The presence of antimicrobial compounds that are therapeutically potent against MDR bacteria was confirmed in W. fruticosa. The crude leaf extract showed no host toxicity with human lymphocytes; the n-butanol fraction of the extract was the most suitable bioactive fraction. The terpenes isolated were: 5-methyl-2-(1-methylethyl) phenol, 2-methoxy-4-(2-propenyl) phenol, 2,6-octadien-1-ol, 3,7-dimethyl-(E)-2,6-octadienal, 3,7-dimethylcyclohexanol, and cyclohexanol, 2-methylene-5-(1-methylethenyl) which were reported to have specifically antimicrobial activity.
PMCID: PMC4225590  PMID: 25389517
gas chromatography–mass spectrometry analysis; human lymphocytes; multidrug-resistant bacteria; Woodfordia fruticosa
24.  Polybrominated biphenyl toxicosis in rats fed an iodine-deficient, iodine-adequate, or iodine-excess diet. 
Young male Sprague-Dawley rats were fed 0, 1, 10, or 100 ppm of polybrominated biphenyls (PBB) in iodine-deficient, iodine-adequate (0.2 ppm), or iodine-excess (1000 ppm) diets. Six rats in each of the 12 groups were killed at 30 days and the remaining six in each group at 60 days. Growth rates were similar in all rats fed diets containing 0, 1, or 10 ppm PBB but were slower from 30 to 60 days in rats given 100 ppm PBB. Results of routine hematologic examinations and urinalyses were essentially normal. Although liver weights were substantially increased by PBB, the smallest increases were in rats fed an iodine-deficient diet. Thyroid weights were increased by iodine deficiency and by 10 and 100 ppm PBB. Electropherograms of serum proteins, serum lipoproteins, and LDH isozymes at 60 days from rats given PBB indicated hepatic alterations, but changes were least dramatic in rats fed an iodine-deficient diet plus PBB and most severe in rats fed iodine-excess diets plus PBB. Hepatic lesions were basically similar to those previously described except that bile duct proliferation was seen at 60 days only in rats fed an iodine-deficient diet and 100 ppm PBB. Histologic changes in thyroid glands were associated with iodine deficiency and with PBB. The iodine-excess diet plus 100 ppm PBB induced squamous metaplasia of respiratory bronchiolar epithelium. These results indicate interrelationships between PBB and iodine which may affect the toxicosis caused by PBB.
PMCID: PMC1637452  PMID: 209997
25.  Radioimmunoassay of the binding protein for vitamin D and its metabolites in human serum: concentrations in normal subjects and patients with disorders of mineral homeostasis. 
Journal of Clinical Investigation  1976;58(5):1217-1222.
A radioimmunoassay for the binding protein for vitamin D and its metabolites (DBP) has been developed. Suitable rabbit anti-DBP antiserum was elicited after primary and one booster injection. Anti-DBP antisera, as well as antigroup-specific component antisera, produced a single, monospecific line of percipitation when reacted against purified DBP and human serum. DBP was iodinated with 125I and 125I-DBP was purified by gel filtration on Sephadex G-200. Binding of 125I-DBP by 20 nl of rabbit anti-DBP antisera was approximately 50% and was sharply competed for by 0.4-4.0 ng of DBP standard. Displacement of 125I-DBP by human serum dilutions or standard DBP gave identical curves, and only weak competition was observed with old and new world primate sera. Apo- and holo-DBP possessed indistinguishable immunoreactivity. The assay detects DBP in 1-10 nl of human serum with reasonable accuracy and with reasonable intra- and interassay precision. The mean serum concentration (+/- SEM) for a group of 40 normal adults was 525 +/- 24 mug/ml and no sex difference was observed. Higher levels were found in sera from pregnant women and women receiving oral contraceptives, and decreased concentrations were observed in premature cord and hypoproteinemic sera. No significant correlation between serum DBP levels and serum 25-hydroxycalciferol levels was found, and the DBP content of sera from vitamin D-deprived and vitamin D-treated subjects was indistinguishable from that of normal adults. DBP accounts for 6- of the alpha globulin in normal human serum. Considering the normal serum content of the parent vitamin and its metabolites to be approximately 0.1-0.2 mum, these immunoassay data confirm previous saturation analyses of human serum antiricketic sterol-binding capacity and suggest that greater than 95% of DBP circulates as the apoprotein under normal conditions.
PMCID: PMC333290  PMID: 1086857

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