Increased hepatic lipase activity and increased direct removal of very-low-density lipoprotein remnants in Watanabe heritable hyperlipidaemic (WHHL) rabbits treated with ethinyl oestradiol.

We studied the effects of ethinyl oestradiol on the serum concentrations and metabolism of very-low- and low-density lipoproteins (VLDL and LDL) in Watanabe heritable hyperlipidaemic (WHHL) homozygous rabbits, an animal model for familial hypercholesterolaemia. The results were compared with those in untreated homozygotes as well as in heterozygotes treated or not with ethinyl oestradiol. The gain in body weight was similar in all groups. Treatment with ethinyl oestradiol resulted in the homozygotes in an approx. 80% decrease in the concentrations of lipids and apoprotein B in the d less than 1.019 lipoprotein fraction; those in the LDL fraction did not change. In the heterozygotes, basal serum lipids and apoprotein B levels in the d less than 1.019 fraction were low; ethinyl oestradiol treatment especially affected the LDL fraction (cholesterol -84%, apoprotein B -64%). Turnover experiments with 125I-labelled VLDL revealed that, on treatment with ethinyl oestradiol, the fractional catabolic rate in homozygous rabbits increased 2-fold. The secretion rates of lipids and protein in the d less than 1.019 fraction as estimated after injection of Triton WR-1339 was not decreased. In homozygotes and heterozygotes increases in post-heparin hepatic lipase activity of 62 and 80% respectively were observed, with no changes in lipoprotein lipase activity. We conclude that ethinyl oestradiol induces in homozygous WHHL rabbits a direct removal of VLDL and VLDL remnants from the plasma, apparently due to an increase in hepatic lipase activity.     

Kidney and liver metallothioneins in rats after administration of an organic compound

Intubation of rats with alpha-mercapto-beta-(2-furyl)-acrylic acid (MFA) for 5 days at 50 mg/kg caused a 7-fold increase in kidney copper concentration, a 2-fold increase in kidney zinc concentration, and a 20% increase in liver zinc concentration. The proteins which bound the increased metals were purified and identified as metallothioneins by their amino acid compositions. Two isoforms were isolated from each organ. Renal thioneins appeared identical to counterpart hepatic apoproteins, but the former bound Cu and Zn in a 2:1 mole ratio and the latter bound only Zn. Kidney contained over 10 times more metallothionein per g of tissue than did liver. In rats previously administered MFA, injection of cadmium sulfate resulted in rapid displacement of liver metallothionein-bound Zn by Cd under conditions where minimal metallothionein was found in Cd-dosed animals not administered MFA. We conclude that MFA induces metallothionein biosynthesis in kidney and liver of normal rats; this is a novel effect for an organic compound.     

Developmental changes of selected minerals in Zucker rats

Effects of obesity and age on copper, iron, zinc, sodium, potassium, and protein were compared in liver, kidney, brain, and muscle of obese (fa/fa) and nonobese (non-fa/fa) male Zucker rats. Blood plasma cerulopasmin, copper, zinc, sodium, and potassium were also determined. Mean brain weight of fa/fa rats was less than that of non-fa/fa rats at 12 weeks of age; mean brain protein concentration was greater in fa/fa than in non-fa/fa at 5 and 12 weeks of age. At 18-19 days of age, mean sodium concentration (mg/g protein) in liver of fa/fa was less than that of non-fa/fa. At 5 weeks of age, mean copper concentration (microgram/g protein) in kidney was greater in fa/fa. Mean total copper, iron, zinc, sodium, and potassium in liver and kidney were greater in fa/fa than in non-fa/fa at 5 weeks because of the larger livers and kidneys of fa/fa. Mean concentrations of copper, zinc, sodium, and potassium per gram of brain protein were slightly (6-10%) less in fa/fa than in non-fa/fa at 5 weeks. By 12 weeks, mean concentrations of copper in liver, kidney, (tibialis) muscle, and blood plasma, ceruloplasmin in blood plasma, zinc in liver and muscle, iron in muscle, and sodium in liver were greater in fa/fa than in non-fa/fa. However, total amount of each mineral in muscle at 12 weeks was less in fa/fa than in non-fa/fa because of the smaller mean muscle weight of fa/fa. Mean concentrations of copper and zinc in brain and of iron in liver and brain were less in fa/fa than in non-fa/fa at 12 weeks. The major age-related changes in fa/fa that were not observed in non-fa/fa were large increases in liver and kidney copper between 5 and 12 weeks of age. It seems that the abnormal mineral metabolism is a consequence of the obesity, but the mechanisms are not identified.     

The distribution of zinc and copper in plasma, erythrocytes and erythrocyte membranes of rainbow trout (Salmo gairdneri)

1. The zinc and copper concentration of plasma was determined in rainbow trout, lake trout, walleye and whitefish. 2. These fish had mean plasma zinc concentrations ranging from 9.3 to 15.1 ppm and copper concentrations from 0.6 to 1.3 ppm. 3. In rainbow trout, the concentration of zinc and copper is greater in the erythrocyte membrane than in the total erythrocyte. 4. Ultrafilterable plasma zinc and copper concentration in rainbow trout was determined to be 0.03 and 0.019 ppm, respectively. 5. Dialysis of rainbow trout plasma against 20 mM EDTA results in removal of 99% of the zinc and 88% of the copper from plasma proteins.     

The regulation of the biologically available fractions of oestradiol and testosterone in plasma

The albumin bound fractions of oestradiol and testosterone have been measured in samples of plasma obtained over a 24 h period from women with breast cancer or polycystic ovarian disease and from pre- and postmenopausal control subjects and related to plasma levels of free fatty acids. For most subjects changes in the fraction of oestradiol bound to albumin were related to changes in plasma levels of free fatty acids. A significant decrease in the albumin bound testosterone fraction during the night was associated with increased plasma levels of cortisol.     

Influence of egg production on zinc, copper and iron metabolism in the turkey hen (Meleagris gallopavo)

1. Serum concentrations of zinc, copper, iron, calcium and protein increased significantly with the onset of egg production in turkey hens. 2. Hepatic concentrations of zinc, copper and iron declined significantly with the onset of egg production, whereas the total hepatic content of zinc and copper increased significantly. 3. When plasma from laying and nonlaying hens was subjected to gel-permeation column chromatography, a high molecular weight protein fraction was detected in laying but not in nonlaying plasma which bound significant amounts of zinc, copper and iron and which coeluted with purified vitellogenin. 4. Vitellogenin purified from the plasma of laying hens is a metalloprotein which upon analysis was found to contain 0.54, 0.09 and 0.72 micrograms of zinc, copper and iron, respectively, per mg of protein.     

The effect of ethinyl oestradiol on calcium and bone metabolism in peri- and postmenopausal women

The effects of ethinyl oestradiol on plasma and urinary calcium and other indices of bone turnover have been compared in peri- and postmenopausal women. In postmenopausal women ethinyl oestradiol caused significant decreases in the fasting plasma total and ionised calcium and phosphate concentrations, in alkaline phosphatase activity and in the fasting urinary Ca/Cr and OHPr/Cr ratios. Similar, but less marked, effects were observed in peri-menopausal women, with significant decreases in the fasting plasma phosphate and urinary Ca/Cr ratio. Small decreases in the plasma total and ionised calcium concentrations, in alkaline phosphatase activity and in the urinary OHPr/Cr ratio also occurred but were not statistically significant in our sample. Ethinyl oestradiol therefore appears to reduce bone loss in peri- as well as post-menopausal women, at least in the short term, but the effect is less pronounced in the former group.     

Tissue-and metal-specific effects of thiolacrylic acids in rats

Kidney copper increased 12- to 18-fold above the normal level in rats administered alpha-mercapto-beta-(2-furyl)acrylic acid (MFA). Kidney zinc increased twofold; plasma zinc increased more than 10-fold and liver zinc increased 30–50%. No other changes in copper, iron, and zinc concentrations were found in these tissues or in bone, brain, heart, lung, skeletal muscle, spleen, or testis. Related compounds produced similar effects, although MFA and its disulfide were the most potent of the compounds tested. These increases in tissue copper and zinc were largely complete after 2–5 d of daily administration of compound. Increased plasma zinc returned toward normal with a half-life of 1.0 d for the process, after dosing was ended; albumin was identified as the species binding the excess zinc in plasma. Kidney copper and zinc, which had increased in the ratio of 3 Cu/Zn, returned to normal levels after dosing was stopped with half-lives of 2.1–2.5 d. Consistent with the observations of highly tissuespecific effects of MFA, copper and zinc balances over 8 weeks of trials were found to be not greatly affected by administration of the compound. Thus, it was not established whether excess metal in affected organs derived from enhanced retention of dietary metal or redistribution from other tissues. Kidney copper and zinc and serum zinc increased even in zinc-deficient rats administered MFA.     

Involvement of the liver in the regulation of tryptophan availability. Possible role in the responses of liver and brain to starvation

The concentrations of free and total (free plus albumin bound) tryptophan were measured in plasma of blood taken from the portal vein, hepatic vein and abdominal aorta of male rats, fed, and starved for one and three days. Liver and brain tryptophan concentrations were measured in similar groups of rats.On starvation, there was an increase in arterial plasma free tryptophan concentration which took place peripherally and was paralleled by an increase in brain tryptophan. In both the fed and starved rats, the portal vein concentrations of free tryptophan were high and as the blood flowed through the liver they were reduced to relatively low levels not directly related to the arterial values. All these changes were due to alterations in degree of binding of tryptophan to plasma albumin.The measurements of plasma total tryptophan concentrations showed that postabsorptively and during starvation there was a net uptake of tryptophan by the peripheral tissues (which included brain), but no overall fall in plasma concentration. At the same time, there was a net release from the liver, and to a lesser extent from the portal-drained tissues. The released tryptophan largely entered the albumin bound plasma pool. Accompanying the hepatic output was a fall in tryptophan concentration in the liver which was apparently caused by altered cell membrane transport.The results suggest (1) that the liver protects the brain from the high free tryptophan level in portal blood, (2) that the availability of tryptophan to the brain is maintained postabsorptively and during starvation by hepatic output into the albumin bound pool and (3) that this release of tryptophan from the liver and the fall in intracellular tryptophan concentration are initiated by altered membrane transport. The pattern of changes is consistent with a role for tryptophan in the mediation of changes in liver protein synthesis and gluconeogenesis and cerebral serotonin turnover on starvation.     

The effects of androgens and cortisol on the in vivo metabolism of oestradiol

We have examined the effect of co-administration of dehydroepiandrosterone sulphate, 5-androstenediol or cortisol on the metabolic clearance rate of oestradiol (MCR-E2) and conversion of oestradiol to oestrone (CRE2E1). Previous studies have shown that these androgens influence the metabolism of oestradiol in vitro while cortisol alters the distribution of oestradiol in plasma. The MCR-E2 and CRE2E1 were measured after 2.5 and 5 h of [3H]oestradiol infusion with co-infusion of androgen or cortisol starting after 2.5 h of tracer infusion. For one subject who did not receive co-infusion of another steroid no significant change in MCR-E2 or CRE2E1 occurred over the 5-h period. For other subjects, however, the MCR-E2 decreased by 18 +/- 7% (mean +/- SD) while the CRE2E1 increased by 45 +/- 12%. It is possible that these results are due to: changes in the distribution of oestradiol in plasma; differences in the metabolism of oestradiol bound to albumin or SHBG, or an effect of androgens or cortisol on the uptake of [3H]oestradiol by the liver.     

Plasma binding of oestradiol in progesterone-treated rats

Progesterone treatment of female rats causes an increase in body weight possibly via suppression of oestradiol secretion. This study was carried out to investigate the effect of progesterone on the non-protein bound and hence presumably biologically active fraction of oestradiol. Oestradiol binding to plasma proteins was studied in female Wistar rats during the oestrous cycle and after 12 days of treatment with progesterone (5 mg/day). There was no change in either the unbound fraction of oestradiol or plasma albumin concentrations during the oestrous cycle. Plasma oestradiol concentrations in progesterone-treated rats were similar to those seen during dioestrus, as were the degree of oestrogen binding and the plasma albumin concentrations. Although it was not feasible to calculate unbound concentrations, these results suggest that the increased body weight seen in progesterone-treated rats, and also during pregnancy, may be a result of suppression of unbound oestradiol concentrations to levels similar to those occurring during dioestrus.     

Use of a single-injection tissue-sampling technique to study steroid uptake by the decidualized endometrium of the mouse

A tissue-sampling paired-tracer method was used to investigate the effect of plasma proteins on uptake by the decidualized endometrium of [3H]progesterone, [3H]oestradiol and [3H]corticosterone. When injected arterially in protein-free Ringer, the extraction of progesterone and oestradiol was 100%, while that of corticosterone was only 60%. The addition of 4% albumin or injection in mouse plasma resulted in significant decreases in progesterone extraction to about 80% and 65% respectively. Injection in pregnant guinea-pig plasma reduced progesterone extraction further (to 33%). While neither 4% albumin nor mouse plasma had any significant effect on the uptake of oestradiol, neonatal rat plasma reduced oestradiol extraction to 40%. These results are consistent with high-affinity binding proteins having a limiting effect on the availability of steroids to target tissues.     

Effects of Albumin, Amino Acids and Clofibrate on the Uptake of Tryptophan by the Rat Brain

Abstract: The influences of total tryptophan concentration, albumin binding and amino acid competition on the rate of tryptophan influx into rat brain were compared using a single-pass injection technique with tritiated water as a freely diffusible reference. Omission of 3% bovine albumin from a bolus containing tryptophan in Krebs–Ringer bicarbonate buffer injected into the carotid artery increased non-albumin bound (free) tryptophan concentration threefold but tryptophan uptake by only 35% and 30% into forebrain and hypothalamus, respectively. However, tryptophan uptake from injected rat plasma was more markedly elevated when free tryptophan concentration was raised. Thus, when free tryptophan was doubled, but total tryptophan unchanged, by in vitro addition of clofibrate to a plasma bolus, uptake was increased by 53% and 28% into forebrain and hypothalamus respectively. When clofibrate was injected in vivo so that plasma total tryptophan concentration was decreased by 45% but neither free tryptophan nor competing amino acid concentrations were altered, then uptake from a bolus of the rat's own plasma was unchanged. Addition of competing amino acids at physiological concentrations to tryptophan in Krebs-Ringer buffer significantly reduced tryptophan influx into both brain regions, but did not increase the effect of albumin binding. The results indicate that tryptophan uptake into rat forebrain is substantially influenced by albumin binding and competition from other amino acids, but that hypothalamic uptake is less influenced by these factors.     

Obesity and Elevated Plasma Leptin Concentration in oMT1A‐o Growth Hormone Transgenic Mice

OBERBAUER, A. M., JONATHAN A. RUNSTADLER, JAMES D. MURRAY, AND PETER J. HAVEL. Obesity and elevated plasma leptin concentration in oMT1A‐o growth hormone transgenic mice. Objective: This study was undertaken to evaluate plasma leptin concentration in the regulatable ovine metallothionein‐ovine growth hormone (oMT1a‐oGH) transgenic (TG) mouse model of obesity. Research Methods and Procedures: Transgene stimulus (zinc) was provided at 21 days of age to male and female wild‐type (WT) and TG mice. Plasma leptin concentrations were measured by radioimmunoassay at 42, 63, 84, and 105 days of age and from inactivated TG mice at 84 and 105 days. Results: WT and TG mice did not differ significantly in plasma leptin concentration at any of the ages examined (42, 63, 84, and 105 days), although females showed consistently higher plasma leptin concentrations than males regardless of genotype throughout the duration of the study. Male and female TG mice in which the transgene was inactivated at 63 days had a 1.5‐fold to 3.5‐fold increase in plasma leptin concentration over WT mice and continuously activated TG mice at 84 and 105 days of age. The elevated plasma leptin concentration seen in the inactivated TG mice at 84 and 105 days of age reflects the >300% increase in white adipose tissue seen in this model and correlated with all adipose depot weights and overall body lipid at these later ages. When plasma leptin was expressed per gram of total body fat, the leptin adjusted for body lipid was significantly higher in WT mice than either continuously activated TG or activated and then inactivated TG groups. Discussion: The inactivated TG mice in this study had higher plasma leptin levels with increasing total body adiposity, but the relative proportion of circulating leptin, on a total body lipid basis, was reduced when compared with the WT mice. This reduction was also observed in activated TG mice at the older ages. Although the absolute levels of circulating leptin were elevated in the inactivated TG animals, the amount of leptin produced per gram of fat was lowered. With the inactivation of the transgene, the leptin remained depressed after the removal of the elevated growth hormone. This represents a potential explanation for the ensuing hypertrophy of the fat depots and the abnormal phenotypic response of inactivated TG mice to elevated plasma leptin concentrations resulting in the development of obesity.     

Fractionation of Soluble Copper- and Zinc-Binding Proteins From Cattle Liver

The distribution of copper and zinc among soluble proteins in liver from normal slaughter cattle was examined after gel filtration of the proteins. Gopper- and zinc-binding proteins were mainly separated into three fractions. Varying amounts of zinc were eluted in a fourth fraction of molecular weight less than 2,000. A clear relationship was noted between the amount of copper bound to the low molecular weight fraction (m.w. ~ 10,000) and the total liver zinc concentration. The high molecular weight protein fraction (m.w. > 65,000) dominated in liver with zinc concentrations below 40 µg/g wet weight and total copper concentrations from 16 to 240 µg/g, while in liver with zinc concentrations above 40 µg/g and copper concentrations ranging from 20 to 107 µg/g, the low molecular weight metallothionein-like fraction dominated.     

Oestrogen and nuclear binding sites. Determination of specific sites by [3H]oestradiol exchange

A method was developed for the determination of the number of specific oestradiol-binding sites in the nuclear fraction of oestrogen-sensitive tissues. The method is based on the exchange of [(3)H]oestradiol with non-labelled oestradiol that is bound to nuclear binding sites. The number of specific nuclear binding sites after the injection of 2.5mug of oestradiol, an amount sufficient to saturate all binding sites, is 1.6-1.7pmol per immature uterus. The number of sites occupied after an injection of physiological amounts of oestradiol (0.1mug) was 0.46pmol. The injection of oestradiol results in an increased number of nuclear binding sites in uterus and vagina, but has no effect on kidney or muscle. Injections of testosterone or progesterone failed to increase the number of uterine nuclear binding sites. This method permits an evaluation of the number of oestradiol-binding sites in the nuclear fraction of various tissues as a function of either endogenous oestradiol or non-labelled oestradiol administered by injection.     

The tissue disposition and urinary excretion of cadmium, zinc, copper and iron, following repeated parenteral administration of cadmium to rats.

The effect of repeated parenteral administration of cadmium (0.75, 1.5 and 3.0 mg/kg) on tissue disposition and urinary excretion of cadmium, zinc, copper and iron has been studied in the male rat. Cadmium, zinc and copper accumulated in liver and kidney, but the concentration of iron did not alter significantly. The kidney weight relative to body weight showed a dose-related increase in weight of 25--65%. Excretion of cadmium in the urine increased directly with dosage and the increase was most significant when kidney damage had probably occurred. Administration of cadmium also resulted in dose-related increases in the urinary excretion of zinc, copper and iron. The cadmium concentration of blood increased with dosage of cadmium, and the plasma concentrations of zinc and copper were also raised but plasma iron concentration was diminished.     

Oestrogenic sensitivity of rat uterine secretion.

Ovariectomized adult rats with closed uteri were treated for 7 days with different oral and s..c. doses of oestradiol, oestrone, oestriol and ethinyl oestradiol. All treatments elicited the production of uterine fluid and the potencies of oestrogens were related to the amount of fluid secreted. Ethinyl oestradiol and oestradiol displayed similar activity when given s.c. A daily dose of 0-003 mg oestradiol/kg resulted in about 700 mg fluid. Oestrone was 3-10 times and oestriol about 100 times less active. Orally, ethinyl oestradiol was the most potent substance and 700 mg secretion was obtained with a dose of 0-03 mg/kg daily. Oestradiol was about 30 times, oestrone about 100 times and oestriol 50 times less active than ethinyl oestradiol by this route. The viscosity of the secretion was unaffected, remaining between 1-6 and 2-4 cP. The pH of the fluid did not change, but that of the uterine lumen diminished slightly. These effects of oestrogens were associated with an increase in the weight of the empty uterus and a decrease in body weight.     

Computer simulation of Zn(II) speciation and effect of Gd(III) on Zn(II) speciation in human blood plasma

The speciation and distribution of Zn(II) and the effect of Gd(III) on Zn(II) speciation in human blood plasma were studied by computer simulation. The results show that, in normal blood plasma, the most predominant species of Zn(II) are [Zn(HSA)] (58.2%), [Zn(IgG)](20.1%), [Zn(Tf)] (10.4%), ternary complexes of [Zn(Cit)(Cys)] (6.6%) and of [Zn(Cys)(His)H] (1.6%), and the binary complex of [Zn(Cys)₂H] (1.2%). When zinc is deficient, the distribution of Zn(II) species is similar to that in normal blood plasma. Then, the distribution changes with increasing zinc(II) total concentration. Overloading Zn(II) is initially mainly bound to human serum albumin (HSA). As the available amount of HSA is exceeded, phosphate metal and carbonate metal species are established. Gd(III) entering human blood plasma predominantly competes for phosphate and carbonate to form precipitate species. However, Zn(II) complexes with phosphate and carbonate are negligible in normal blood plasma, so Gd(III) only have a little effect on zinc(II) species in human blood plasma at a concentration above 1.0×10⁻⁴ M.     

Metal-binding functions and antioxidant properties in human thyroid gland under iodine deficient nodular colloidal goiter

Copper and zinc levels in the tissue of thyroid gland (TG) and their metal-binding proteins metallothioneins (MT) as well as state of the antioxidant system in persons that had no thyroid disease and patients with endemic iodine deficiency nodular colloidal goiter has been investigated. In the patients with thyroid disease, oxidative damage was indicated despite elevated levels of MT-SH and glutathione, and elevated copper and decreased zinc concentration in TG tissue. MTs partly bound the excess of copper but its concentration in the unbound to MT form was two-fold compared to the control value.