Changes in renal tri-iodothyronine and thyroxine handling during fasting.

OBJECTIVE: Liver handling of thyroid hormones (TH) has been known to alter significantly during fasting. This study investigates whether renal handling of TH is also changed during fasting. METHODS: We measured urinary excretion rates and clearances of free tri-iodothyronine (T(3)) and free thyroxine (T(4)) in healthy subjects prior to and on the third day of fasting. RESULTS: During fasting, both mean T(3) and T(4) urinary excretion decreased significantly to a mean value of 42% of control. Also, total and free (F) serum T(3) concentrations declined significantly, but serum T(4) did not change. Both FT(3) and FT(4) clearance decreased significantly during fasting (62% and 42% of control). The fasting-induced decrease in uric acid clearance correlated well with the decrease in FT(3) clearance (r=0.94; P<0.001). Serum concentrations of non-esterified fatty acids (NEFA) were significantly elevated during fasting. CONCLUSIONS: The findings cannot be fully explained by the fasting-induced decrease in serum T(3), and are in accordance with inhibition of uptake of T(3) and T(4) at the basolateral membrane of the tubular cell. This inhibition may be caused by a decreased energy state of the tubular cell and by other factors such as ketoacidosis and/or increased NEFA concentrations during fasting.

Chromogranin A as serum marker for neuroendocrine neoplasia: comparison with neuron-specific enolase and the alpha-subunit of glycoprotein hormones.

Chromogranin A (CgA) is gaining acceptance as a serum marker of neuroendocrine tumors. Its specificity in differentiating between neuroendocrine and nonneuroendocrine tumors, its sensitivity to detect small tumors, and its clinical value, compared with other neuroendocrine markers, have not clearly been defined, however. The objectives of this study were to evaluate the clinical usefulness of CgA as neuroendocrine serum marker. Serum levels of CgA, neuron-specific enolase (NSE), and the alpha-subunit of glycoprotein hormones (alpha-SU) were determined in 211 patients with neuroendocrine tumors and 180 control subjects with nonendocrine tumors. The concentrations of CgA, NSE, and alpha-SU were elevated in 50%, 43%, and 24% of patients with neuroendocrine tumors, respectively. Serum CgA was most frequently increased in subjects with gastrinomas (100%), pheochromocytomas (89%), carcinoid tumors (80%), nonfunctioning tumors of the endocrine pancreas (69%), and medullary thyroid carcinomas (50%). The highest levels were observed in subjects with carcinoid tumors. NSE was most frequently elevated in patients with small cell lung carcinoma (74%), and alpha-SU was most frequently elevated in patients with carcinoid tumors (39%). Most subjects with elevated alpha-SU levels also had elevated CgA concentrations. A significant positive relationship was demonstrated between the tumor load and serum CgA levels (P < 0.01, by chi 2 test). Elevated concentrations of CgA, NSE, and alpha-SU were present in, respectively, 7%, 35%, and 15% of control subjects. Markedly elevated serum levels of CgA, exceeding 300 micrograms/L, were observed in only 2% of control patients (n = 3) compared to 40% of patients with neuroendocrine tumors (n = 76). We conclude that CgA is the best general neuroendocrine serum marker available. It has the highest specificity for the detection of neuroendocrine tumors compared to the other neuroendocrine markers, NSE and alpha-SU. Elevated levels are strongly correlated with tumor volume; therefore, small tumors may go undetected. Although its specificity cannot compete with that of the specific hormonal secretion products of most neuroendocrine tumors, it can have useful clinical applications in subjects with neuroendocrine tumors for whom either no marker is available or the marker is inconvenient for routine clinical use.

High tissue factor concentration in the omentum, a possible cause of its hemostatic properties.

The hemostatic properties of the pedicled omentoplasty turned out to be helpful in difficult hemorrhages in extensive surgery. As suggested by others, a high concentration of tissue factor (TF) in the omentum could be responsible for this favourable property. The authors investigated the nature of that property in 11 patients who underwent laparotomy. In omentum and striated muscle (controls) the TF-concentrations in both tissues were estimated by the ELISA method. A significant difference between TF-concentration in omentum and striated muscle could be demonstrated.

Investigation of type I and type III iodothyronine deiodinases in rat tissues using N-bromoacetyl-iodothyronine affinity labels.

In the present study the hypothesis was tested that N-bromoacetyl-3,3',5-[125I]triiodothyronine (BrAc[125I]T3) is a useful affinity label for both type I and type III iodothyronine deiodinases (ID-I and ID-III). Therefore, the microsomal fractions of various rat tissues were tested for ID-I and ID-III activities, and microsomal proteins were labeled with BrAc[125I]T3 and analyzed by SDS-PAGE. In agreement with previous observations, high ID-I activities were found in liver, kidney and thyroid, and high ID-III activities in brain, in particular fetal brain, and placenta. SDS-PAGE of BrAc[125I]T3-labeled microsomes showed a prominent radioactive approximately 27 kDa protein (p27) in liver, kidney and thyroid, which was previously identified as ID-I, and a approximately 32 kDa protein (p32) in brain, in particular fetal brain, and placenta. A good correlation was found between the affinity labeling of p32 and the inactivation of ID-III by BrAcT3, suggesting that p32 represents ID-III or a subunit thereof. After treatment of microsomes with 0.05% deoxycholate or carbonate buffer (pH 11.5) p32 was still labeled by BrAc[125I]T3, indicating that p32 is a transmembrane protein. Although 3,3',5'-triiodothyronine (rT3) is not a substrate for ID-III, p32 was readily labeled with BrAc[125I]rT3. Labeling of p32 in rat brain microsomes by BrAc[125I]rT3 was not affected by addition of 100 microM unlabeled thyroxine (T4) or T3, whereas deiodination of [125I]T3 by ID-III was inhibited by 91 and 96% in the presence of 1 microM T4 and T3, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation of a new tumour marker in patients with non-small-cell lung cancer: Cyfra 21.1.

The Cyfra 21.1 assay is a newly developed test which measures in serum a fragment of cytokeratin 19. We evaluated this marker in 212 patients with non-small-cell lung cancer (NSCLC), predominantly stage 3a-b and 4, and compared it with three other markers: carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC) and tissue polypeptide antigen (TPA). Sensitivities for Cyfra 21.1, TPA, CEA and SCC (using cut-off levels corresponding to a 95% specificity for benign lung diseases) were 40%, 40%, 42% and 19% respectively. The sensitivity of CEA was significantly higher in patients with adenocarcinomas compared with the other three markers, while the sensitivity of Cyfra 21.1 and TPA was significantly higher in patients with squamous cell carcinomas. The value of Cyfra 21.1 for monitoring disease during chemotherapy could be evaluated in 23 patients with squamous cell carcinomas. When the cases of lead time were included a concordance between clinical evaluations according to WHO response criteria and evaluations according to changes in the marker levels of 74% was found. The criteria defined for marker response were a 65% decrease in the marker level for a partial response and a 40% increase for progressive disease. In particular, increasing levels of this marker indicated usually disease progression. In conclusion, Cyfra 21.1 is a useful serum marker for patients with NSCLC, especially for disease monitoring of patients with squamous cell carcinoma during and after chemotherapy.

Prognostic significance of tissue polypeptide-specific antigen (TPS) in patients with advanced non-small cell lung cancer.

In this study, we evaluated the prognostic value of the tumour marker, tissue polypeptide-specific antigen (TPS), in 203 patients with non-small cell lung cancer (NSCLC), and related this to several other known prognostic factors. TPS was significantly correlated with lactate dehydrogenase (LDH), gamma-glutamyltranspeptidase and alkaline phosphatase, and the median level of TPS in patients with stage 4 disease was significantly higher as compared to stage 3A and 3B disease. In the univariate analysis, performance status, stage of disease, LDH, alkaline phosphatase, a histology of undifferentiated large cell carcinoma and TPS all had a statistically significant association with survival. Multivariate analysis showed that stage of disease, performance status, histology and TPS were the most important prognostic factors. TPS has prognostic significance for survival in patients with advanced NSCLC, independent from performance status and stage of disease.

Automated enzymatic methods for creatinine measurement with special attention to bilirubin interference.

Four enzymatic methods for creatinine measurement were evaluated on a DuPont Dimension automatic analyzer. Biomed Creatinine-Duo-UV (BIO) and Raichem Creatinine Reagent Enzymatic (RAI) start creatinine breakdown with creatinine iminohydrolase (EC 3.5.4.21) resulting in the formation of NH4+. The Boehringer Mannheim Creatinine PAP (BM1) and SopaChem Creatinine (SOP) start the breakdown of creatinine with creatininase (EC 3.5.2.10) which yields creatine. In order to reduce bilirubin interference, the BM1 method was modified to contain K4Fe(CN)6. This substance was added with reagent 1 (BM2) or with reagent 2 (BM3). All the enzymatic creatinine methods tested displayed good linearity for concentrations up to at least 1000 mumol/l. The BIO, BM3, RAI and SOP methods showed good stability of test outcome for the tested period of a week. The outcome of the BM1 and BM2 method increased continually with time. Only the results of the RAI method were diminished by the presence of lipids. The BM1, BM2, BM3 and SOP methods showed no interference with haemoglobin, whereas this increased the outcome of the BIO method and slightly decreased the results of the RAI method. Using spiked human albumin solutions it was found that the BIO, BM2, BM3 and RAI methods displayed good resistance to interference by bilirubin or ditauro-bilirubin. The outcome of the BM1 and SOP method was strongly decreased by both bilirubin and ditauro-bilirubin. When creatinine was measured in a panel of sera originating from 100 patients with bilirubin concentrations higher than 50 mumol/l, the obtained results were in close agreement with those found for the spiked human albumin solutions.(ABSTRACT TRUNCATED AT 250 WORDS)

Reaction of the type III iodothyronine deiodinase with the affinity label N-bromoacetyl-triiodothyronine.

The type III iodothyronine deiodinase (ID-III) catalyzes the inner ring deiodination and, thus, the inactivation of the thyroid hormones T4 and T3. ID-III activity in rat brain, rat placenta and embryonic chicken liver is inhibited by the affinity label N-bromoacetyl-T3 (BrAcT3) with an affinity similar to that of T3. Reaction of rat brain and placenta microsomes with BrAc[125I]T3 resulted in the extensive labeling of a 32 kDa protein (p32). However, p32 was also prominently labeled in fetal rat liver microsomes which have no ID-III activity. Labeling of p32 was not influenced by 100 microM substrate analogs or inhibitors of ID-III, some of which completely inhibit ID-III activity at 1 microM. BrAc[125I]T3 labeling of embryonic chicken liver microsomes did not reveal p32 or another protein possibly related to ID-III. In contrast to previous suggestions, it is unlikely that p32 represents ID-III or a subunit thereof.

Impairment of the selenoenzyme type I iodothyronine deiodinase in C3H/He mice.

Type I iodothyronine deiodinase (ID-I) activity is impaired in C3H/He (C3H) mice compared with BALB/c and C57BL/6N (C57) mice. In this study we compared ID-I activity and protein labeling with N-bromoacetyl(-)[125I]T3 (BrAc[125I]T3) or 75Se in liver microsomes of C3H and C57 mice. Hepatic ID-I activity in C3H mice was highly variable with a median of only 18% of that in C57 mice. However, C3H mice had normal serum T4 and T3 levels, although serum reverse T3 was increased. The 28-kilodalton (kDa) ID-I protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of BrAc[125I]T3-labeled microsomes. Labeling of this protein was virtually undetectable in C3H samples with low enzyme activity. ID-I activity in liver microsomes was strongly decreased in Se-deficient mice, which was paralleled by a drastic decrease in BrAc[125I]T3-labeling of the 28-kDa band compared with control mice. Labeling of ID-I with 75Se was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of liver microsomes of [75Se]selenite-injected mice. 75Se labeling of the 28-kDa band was markedly higher in Se-deficient than in control mice and was also markedly higher in C57 than in C3H mice. Finally, liver ID-I messenger RNA (mRNA) was measured on Northern blots using a rat ID-I complementary DNA probe. Messenger RNA levels correlated strongly with ID-I activity, showing a significant decrease in C3H mice. We conclude that in mice, like in rats and humans, ID-I is a selenoprotein. ID-I activity is impaired in C3H mice because of decreased transcription of the ID-I gene or reduced stability of the mRNA.

Species differences in liver type I iodothyronine deiodinase.

The type I iodothyronine deiodinase (ID-I) of liver is an important enzyme for the conversion of the prohormone thyroxine (T4) to the active thyroid hormone 3,3',5-triiodothyronine (T3). Because it is an integral membrane protein of low abundance, purification of ID-I from rat liver has proven to be difficult. We have analyzed ID-I in liver microsomal fractions from various animals to reveal possible species differences and to explore alternative sources for the isolation of the enzyme. ID-I was characterized by enzyme assay with 3,3',5'-triiodothyronine (rT3) as the preferred substrate and by affinity-labeling with N-bromoacetyl-[125I]T3 (BrAc[125I]T3). Labeled ID-I subunit was identified and quantified by SDS-PAGE and autoradiography. The Mr of ID-I in the species investigated varied between 25.7 and 29.1 kDa. Rat and dog liver microsomes had a markedly higher enzyme content than microsomes of human, mouse, rabbit, cow, pig, sheep, goat, chicken or duck liver. Rat liver microsomes showed the highest ID-I activity of all species examined. Turnover numbers for ID-I varied between 264 and 1059 min-1, with rabbit and goat showing the highest values. However, dog liver ID-I displayed an exceptionally low turnover number of 78 min-1. In conclusion, ID-I has similar properties in all species examined with the notable exception of dog.

Characteristics of type III iodothyronine deiodinase.

Type III iodothyronine deiodinase (ID-III) catalyzes the inner ring deiodination of T4 to rT3 and of T3 to 3,3'-T2, representing an important pathway for the inactivation of thyroid hormone. High activities of this "oncofetal" enzyme are found in rat brain, skin and fetal intestine, rat and human placenta, chick embryo liver, monkey hepatocarcinoma cells, human colon carcinoma cells, and tadpole liver. ID-III shows substrate preference for T3 over T4; Km values are approximately 10-fold lower for T3 than for T4 but Vmax values are similar. In contrast to the marked ontogenic pattern of ID-III in different tissues, the enzyme shows little change under pathophysiological conditions, such as fasting and thyroid dysfunction. Brain ID-III activity is decreased in hypo- and increased in hyperthyroidism, but the changes are small. Reaction of brain and placenta microsomes with BrAc 125I-T3 results in extensive labeling of a 32 kDa protein (p32). However, the relationship of p32 with ID-III is not clear, since labeling of p32 is also observed in tissues without ID-III activity and is not inhibited with a large excess of substrate.

Rat liver type I iodothyronine deiodinase is not identical to protein disulfide isomerase.

This study was done to test the recent hypothesis (Boado et al. (1988) Biochem. Biophys. Res. Commun. 155, 1297-1304) that type I iodothyronine deiodinase (ID-I) is identical to protein disulfide isomerase (PDI). Autoradiograms of rat liver microsomal proteins, labeled with N-bromoacetyl-[125I]triiodothyronine (BrAc[125I]T3) and separated by SDS-PAGE, show predominantly 2 radioactive bands of Mr 27 and 56 kDa. Substrates and inhibitors of ID-I inhibited labeling of the 27 kDa band but not that of the 56 kDa band. Treatment of microsomes with trypsin abolished labeling of the 27 kDa protein and destroyed the activity of ID-I but did not prevent labeling of the 56 kDa protein. Following treatment of microsomes at pH 8.0-9.5 or with 0.05% deoxycholate (DOC) PDI content and labeling of the 56 kDa protein were strongly diminished but ID-I activity and labeling of the 27 kDa protein were not affected. The latter decreased in parallel after treatment at pH greater than or equal to 10. Rat pancreas microsomes contain high amounts of PDI but show no ID-I activity. Reaction of these microsomes with BrAc[125I]T3 results in extensive labeling of a 56 kDa protein but no labeling of a 27 kDa protein. Pure PDI (Mr 56 kDa) was readily labeled by BrAc[125I]T3 but showed no deiodinase activity. These results strongly suggest that the 27 kDa band represents (a subunit of) ID-I while the 56 kDa band represents PDI. From these and other data it is concluded that PDI and ID-I are not identical proteins.