Limits of realizing irradiance distributions with shift-invariant illumination systems and finite étendue sources.

When redistributing the light emitted by a source into a prescribed irradiance distribution, it is not guaranteed that, given the source and optical constraints, the desired irradiance distribution can be achieved. We analyze the problem by assuming an optical black box that is shift-invariant, meaning that a change in source position does not change the shape of the irradiance distribution, only its position. The irradiance distribution we can obtain is then governed by deconvolution. Using positive-definite functions and Bochner's theorem, we provide conditions such that the irradiance distribution can be realized for finite étendue sources. We also analyze the problem using optimization, showing that the result heavily depends on the chosen source distribution.

Purification of bovine thyroid peroxidase.

Trypsin-solubilized peroxidase activity from beef subcellular particles was resolved by DEAE-cellulose chromatography into 5 fractions, which contained enzymatically active components that ranged in molecular size from 73,000 to 340,000 daltons. The most active fraction (mol wt, 92,000 by gel filtration) was further purified (59,000-fold overall) by chromatography on hydroxylapatite. This highly purified peroxidase preparation had an absorbance purity ratio (A410:A280) of 0.55 and oxidized iodide (I3-formation) and guaiacol at rates of 300 and 460 micronmol/min/mg, respectively, which were about 3 and 1 1/2 times, respectively, greater than any previously described preparations. The enzyme was contaminated with an inactive protein of equal size. The highly purified peroxidase preparation lost its activity within a few days even when stored at -15 C with iodide. Two of the other DEAE-cellulose fractions contained peroxidase components with estimated sizes (gel filtration) of 73,000, 96,000, and 98,000, which were further purified purified (1,600 and 15,600 fold) on hydroxylapatite. They were 1/4 to 1/40 as active as the highly purified preparation and also became increasingly labile on purification. The remaining two DEAE-cellulose fractions were heterogeneous mixtures of stable peroxidase components whose average molecular sizes (gel filtration) were 220,000, 300,000, and 340,000 daltons, and which were not amenable to further purification on hydroxylapatite. The ratio of guaiacol to iodide activity decreased from 3.0 in the particles to about 1.5 in the highly purified preparations. The turnover numbers of the purest peroxidase component (mol wt. 92,000) for iodide and guaiacol were very similar to those of highly purifed, commericial lacto- and horseradish peroxidases. The pH maxima for iodide oxidation were 7.4, 6.0, and 4.5 for thyroid, lacto-, and horseradish peroxidases, respectively, whereas guaiacol oxidation peaked at pH 7.0-7.8 for all three enzymes. On the basis of these results and the dissimilar molecular sizes reported for trypsin-solubilized thyroid peroxidase by several other investigators, it was concluded that the molecular size is primarily determined by the conditions of proteolysis.

Thyroid hormone metabolism during liver regeneration in rats.

The metabolism of thyroid hormones was studied during the prereplicative period of liver regeneration. After partial hepatectomy, serum thyroxine (T4) and triiodothyronine (T3) levles progressively fell, and reached a nadir at 12 h proportional to the quantity of liver tissue exised. The diminution (60-80%) in serum iodothyronines was related specifically to partial hepatectomy because laparotomy, ether anesthesia, and other stressful surgical procedures did not induce similar changes. At least 3 phenomena appear to be involved: 1) increased utilization and turnover of thyroid hormone by the regenerating liver remmant. 2) diminished hormone secretion by the thyroid gland between 6-12 h after surgery, and 3) a slightly reduced concentration of serum iodothyronine carrier proteins. The results support the concept that the liver participates in the metabolic regulation of T2 and T4 which in turn, control hepatocellular growth. It is suggested, however, that additional unknown factors control increased hepatic thyroid hormone turnover after partial hepatectomy.

A physiological dose of triiodothyronine normalizes cardiac myosin adenosine triphosphatase activity and changes myosin isoenzyme distribution in semistarved rats.

The possibility that the lowering of thyroid hormone levels which occurs in the nonthyroidal illness syndrome results in a hypothyroid state at the cardiac tissue level was examined in semistarved rats. Rats were fed 50% of their normal food intake in the form of a regular diet (R. diet) or low carbohydrate diet (L.C. diet) for 8 weeks. Animals semistarved for 8 weeks on the R. diet lost 42% of their body weight, while plasma T3 and T4 levels decreased by 45-50%. Semistarvation on the L.C. diet resulted in a 19% weight loss and a similar 46-49% decrease in plasma T3 and T4 levels. Ca++-activated myosin ATPase activity declined by 28% and 48% with the R. and L.C. diets, respectively [normal rats myosin ATPase, 1.30 +/- 0.18 mumol Pi/(mg protein . min) (mean +/- SD); semistarvation R diet, 0.93 +/- 0.15; semistarvation L.C. diet, 0.67 +/- 0.15]. The administration of physiological amounts of T3 (0.3 micrograms T3/100 g BW daily) restored the cardiac myosin ATPase activity in both groups. To confirm that the T3 effect was due to a normalization of the thyroid status at the tissue level, hypothyroid animals on a normal diet were injected with 0.3 micrograms T3 for 4 weeks, which resulted in normalization of myosin ATPase activity levels. Thyroidectomized rats receiving daily T3 injections, and when placed on a 50% reduction of food intake for 4 weeks still maintained normal myosin ATPase activity even though they lost 36% of their body weight. Distribution of cardiac myosin isoenzymes was determined by pyrophosphate polyacrylamide gel electrophoresis. In normal cardiac ventricles, myosin isoenzyme V1 predominates and represents 68 +/- 7% (+/- SD) of the total myosin. Semistarvation resulted in a redistribution of myosin isoenzymes so that V3 myosin was the predominant species (53 +/- 3% of the total myosin). The administration of 0.3 microgram T3/100 g BW daily for 4 weeks to semistarved rats reverted myosin isoenzyme distribution to V1 predominance (V1 myosin, 54 +/- 3% of the total myosin). These results indicate that the semistarvation-induced lowering plasma T3 and T4 levels is an important determinant of myosin ATPase activity and myosin isoenzyme distribution. Restoration of myosin ATPase activity to its normal level and return to myosin V1 predominance after T3 administration make it likely that these changes are related to the lowering of thyroid hormone levels.

Polyclonal 3,5,3'-triiodothyronine (T3) antibodies in a euthyroid woman and their effect on radioimmunoassays for T3.

Thyroid function tests in a 17-yr-old euthyroid woman with a slightly enlarged thyroid gland were normal, except for an apparently elevated serum T3 level which was over 8 times greater than the upper limit of normal, when her serum was directly analyzed for T3 by an antibody-coated tube RIA method. When T3 analysis was performed with a RIA procedure that also used serum directly and separated free and bound radiolabeled hormone by polyethylene glycol precipitation, an absurd and conflictingly low result was obtained in which the bound fraction from the patient's serum contained 22% more radiolabeled T3 than the bound fraction from the zero T3 standard. These results suggested the presence of endogenous T3 antibodies which interfered with these RIA procedures. Further studies revealed the presence of IgG-kappa and IgG-lambda polyclonal antibodies that bound T3, but not T4, which accounted for the artifactual elevation in T3. The T3 antibodies were also detected by agarose electrophoresis of serum labeled with tracer quantities of [125I]T3. Analysis for T3 by RIA on Sephadex or RIA of an ethanol extract of the patient's serum both eliminated interference from these endogenous T3 antibodies. The T3 autoantibodies occurred in this patient even though there was no previous history of thyroid medication. Thus, endogenous thyroid hormone-binding antibodies produce artifactually abnormal results with RIA methods that assay serum directly, whereas inactivation and removal of the immunoglobulins by alcohol extraction or Sephadex obviates interference from such proteins.

Amniotic fluid concentrations of iodothyronines and thyrotropin do not reliably predict fetal thyroid status in pregnancies complicated by maternal thyroid disorders or anencephaly.

In this study we report measurements of amniotic fluid (AF) concentrations of iodothyronines and TSH in 69 normal and 16 complicated pregnancies. The latter group included 2 women with untreated hyperthyroidism, 1 patient with untreated hypothyroidism, 5 hyperthyroid patients who received propylthiouracil (3 with Graves' disease, 1 with a multinodular goiter, and 1 with chronic thyroiditis), 3 women with Graves' disease who were hypothyroid after treatment, but who were receiving replacement therapy, and 5 anencephalic pregnancies. AF hormone levels could not be correlated with either maternal or cord serum values, neonatal serum measurements, and/or the clinical status of the infant. AF TSH and T4 levels were markedly elevated in 1 patient with Graves' disease and severe Rh isoimmunization and in 2 pregnancies complicated by anencephaly without identifiable pituitary tissue in the fetus. We conclude that measurements of AF concentrations of thyroid hormones and TSH do not reliably predict fetal or neonatal thyroid status.

Protein-linked iodotyrosines in serum after topical application of povidone-iodine (Betadine).

Markedly elevated serum PBI levels occur after therapy with povidone-iodine (Betadine), an iodine-polyvinylpyrrolidone iodophor. In this study, we investigated the serum iodine compounds from a severely burned patient with normal initial thyroid function tests who was swabbed with Betadine ointment and received daily therapeutic baths in Betadine. Three and 9 days after therapy, his serum contained 93 and 168 micrograms PGI/dl, respectively (normal range, 4-8), while the serum T4 and free T4 index were normal; the serum T3 level, however, was abnormally depressed. Most of the PBI was in albumin, and hydrolysis of the serum proteins with proteases released 35% of the PBI as monoiodotyrosine, 3.2% as diiodotyrosine, 0.01% as T3, and 2.5% as T4, as determined by competitive radioassays, anion exchange, and reversed phase high pressure liquid chromatography. The same concentrations of T4 and T3 were detected before and after hydrolysis. Failure of the proteases to completely hydrolyze iodoalbumin partially explains why all of the PBI was not recovered as iodotyrosines in the serum protein hydrolysates. Povidone-iodine rapidly iodinated tyrosine residues in human serum albumin at pH 7.4 and 37 C in vitro, and the ratio of diiodotyrosine to monoiodotyrosine increased as the molar ratio of povidone-iodine to albumin was increased. It is concluded that the abnormal increase in serum PBI resulted from absorption of the iodophor into the blood where it primarily iodinated albumin and, to a lesser extent, the globulins.

Iodination and the structure of human thyroglobulin.

We have studied human thyroglobulin of extremely low iodine content obtained from a goitrous cretin who had no measurable peroxidase activity in his thyroid gland. Thyroglobulin from these patients is of interest because of the possibility that poorly iodinated thyroglobulin is particularly susceptible to dissociation and proteolysis. In the present study our data indicated that poorly iodinated thyroglobin isolated under conditions inhibiting proteolysis possessed properties similar to normal human thyroglobulin in its secondary, tertiary, and quaternary structures.

Inactivation of intracellular copper-zinc superoxide dismutase by copper chelating agents without glutathione depletion and methemoglobin formation.

The copper chelator N,N'-diethyldithiocarbamate (DDC), is often used to inactivate intracellular copper-zinc superoxide dismutase in erythrocytes. However, in studies with red cells we found that the compound also reacted with oxyhemoglobin to produce oxygen radicals in addition to generating lipid peroxidation products, oxidized N,N'-diethyldithiocarbamate, methemoglobin, and sulfhemoglobin. Moreover, intracellular glutathione was depleted and vital cellular enzymes were susceptible to inactivation. We, and others, have confirmed these findings in nonerythrocytic cell lines. Thus, cells exposed to DDC are severely damaged before studies on the effects of added putative superoxide producing compounds can be performed with them. In this report, we have systematically investigated other copper chelators for their ability to inactivate intracellular copper-zinc superoxide dismutase without producing the deleterious effects mentioned above. Catechol, triethylenetetramine, and tetraethylenepentamine were found to be such agents when erythrocytes were dialyzed in the cold against dilute solutions of these chelators. In addition, with a myeloid leukemic cell line (HL-60), triethylenetetramine inhibited SOD without causing significant GSH oxidation. Examination of the affinity constants of chelators active against purified copper-zinc superoxide dismutase indicated that an affinity binding constant (log K1) between 12.6 and 13.8 was required for the chelator to successfully remove copper from the enzyme.

Fetal hemoglobin: optimum conditions for its estimation by alkali denaturation.

Alkali denaturation is the most commonly used technic to estimate fetal hemoglobin in red blood cells. The Betke and colleagues method (Nature 184: 1959; 1877-78) using cyanmethemoglobin (HiCN) was recommended by an International Committee for Standardization in Hematology for fetal hemoglobin levels between 2 and 40%. We showed that precision with samples containing up to 5% fetal hemoglobin can be considerably improved by measuring the absorbance of HiCN at 420 nm rather than at 540 nm because the molar absorptivity is 10 times greater in the Soret band. To determine optimum conditions for the assay, we studied the kinetics and stoichiometry of conversion of hemoglobin to HiCN with various concentrations and mixtures of ferricyanide, kinetics of alkali denaturation, ammonium sulfate precipitation of denatured HiCN, filtration as opposed to centrifugation for separating denatured HiCN, stability of HiCn in water and KCN solutions, and linearity of absorbance in the Soret band. With our modified procedure, the CV with normal amounts (less than 1%) of fetal hemoglobin improved from 28% to 8.7%. The CVs with 6.8% and 55% fetal hemoglobin were 7.0-4.4% respectively, and linear estimates were obtained with cells containing up to 50% fetal hemoglobin. We conclude that our modified alkali denaturation procedure yields reliable and reproducible estimates of fetal hemoglobin over a wide range of concentrations.

Semi-automated competitive protein binding analysis of serum thyroxine on reusable Sephadex columns and its advantages over radioimmunoassay.

Competitive protein-binding analysis of serum thyroxine on small, reusable, Sephadex columns has been further studied and improved. The improved, semi-automated procedure results in reduced working time and costs. It has also been established that triiodothyronine crossreacts only 1/6 to 1/9 as well as thyroxine, and can be ignored because it represents only about 1/80 of the total serum iodothyronine content. The economic and methodological advantages of the improved method over radioammunoassay and other displacement assays are discussed.

Myeloma immunoglobulin interferes with serum thyroxine analysis by homogeneous enzyme immunoassay.

Myeloma immunoglobulin paraproteins interfere with a homogeneous enzyme immunoassay (EMIT) for serum thyroxine. The EMIT assay failed to detect any hormone in three hyperproteinemic sera from multiple myeloma patients, although thyroxine in these sera was accurately measured by our competitive protein-binding radio-assay on small, re-usable Sephadex columns. The interference was due to turbidity of the paraproteins in the EMIT reaction mixture, resulting in an increased absorbance and a marked underestimation of hormone concentration. Thyroxine was detected (82 to 107% recovery) by the EMIT assay in ethanol extracts of myeloma sera. With 82 other sera there was an excellent correlation (r = 0.985, slope = 0.912, Y intercept (EMIT) = 6.8) of the EMIT assay with our competitive radioassay. Thus, although the EMIT thyroxine assay possesses many desirable features and it is an attractive alternative method to competitive radioassays, its susceptibility to interferences by immunoglobulin paraproteins is a troublesome liability.