Testosterone undecanoate: a useful tool for testosterone administration in rats.

A major obstacle of testosterone (T) treatment in experimental animals is the difficulty of maintaining long-term physiologic/anabolic steady serum levels after exogenous T administration. In two complementary studies we investigated the pharmacokinetic properties of different T formulations in male rats. Study I. Mature male Wistar rats (> 380 g, n = 4 - 7/group) were divided into four treatment groups: (1) sham-operated non orchiectomised (non-ORX) and placebo; (2) orchiectomised (ORX) and subcutaneous testosterone pellets (TP) (15, 25, 75 mg/60 days release or placebo pellets); (3) ORX and a single injection of testosterone undecanoate (TUD) (31, 62.5 or 125 mg/kg body weight subcutaneously (s.c.) or vehicle; (4) ORX and testosterone propionate (Tprop) (10, 20, 40 mg/month) or vehicle as a single injection s.c. Serum T was measured at baseline and in weekly intervals for 4 weeks. Study II. Mature male Wistar rats (180 - 200 g) were randomly assigned to one of 5 experimental groups (n = 5 - 6/group): (1) normal untreated rats (controls); (2) ORX untreated rats, and non-ORX rats receiving one of three treatment options; (3) 250 mg/kg body weight TUD i.m. (TUD 250); (4) 500 mg/kg body weight TUD i.m. (TUD 500); (5) 100-mg testosterone pellet/90 days release s.c. (TP 100). Serum T was measured at baseline and in intervals for 6 weeks after T administration. In both studies, the kinetic profile of TUD showed favourable continuous steady state levels over several weeks. In contrast, testosterone release by subcutaneous pellets resulted in a shorter than expected duration of elevated serum T levels with high inter-individual variability. Tprop administration led to only a short-lasting serum T increase with low serum T levels already 14 days after injection. In conclusion, a single injection of TUD (100 mg/kg body weight s.c.) is effective in inducing physiological testosterone levels in ORX rats for a minimum of four weeks. High dose TUD (500 mg/kg body weight i.m.) given as a single injection results in supraphysiological anabolic testosterone concentrations for up to six weeks in non-ORX rats. TUD was superior to other T release preparations and represents a convenient and effective tool for T administration in experimental animals.

Short-term treatment of rats with high dose 1,25-dihydroxyvitamin D3 stimulates bone formation and increases the number of osteoblast precursor cells in bone marrow.

Using an experimental rat model, this study was undertaken to assess the effects of a short-term application of high dose 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on calcium homeostasis, cancellous bone formation, and numbers of osteoblast precursors in ex vivo bone marrow cultures. For Exp 1 and 2, 6-month-old female rats were sc injected with either 0.2 microg 1,25-(OH)2D3/kg x day or vehicle on days 1, 2, and 3 of the studies. Serum calcium and urinary excretion of calcium were monitored for 12 days in Exp 1. In Exp 2, the rats were ip labeled with five different fluorochromes on days 0, 5, 10, 15, and 20, respectively. Half of the rats in each group were killed on day 7, the rest of the rats were killed on day 24, and the first lumbar vertebrae were processed for histomorphometry. In Exp 3, 0.2 microg 1,25-(OH)2D3/kg BW or vehicle was sc administered to 6-month-old male rats on days 1, 2, and 3, and the number of colony-forming units with the ability to express alkaline phosphatase, to calcify, and/or to synthesize collagen were enumerated sequentially on days 4, 6, 8, 10, 12, and 14 in bone marrow cultures. Short-term 1,25-(OH)2D3 treatment resulted in increased values for serum and urinary calcium during the treatment phase in Exp 1, depressed osteoclast numbers and strongly elevated osteoblast perimeter by day 7, and stimulated mineral apposition rate and bone formation rate in the interval between days 5-15 of Exp 2. Moreover, 1,25-(OH)2D3 administration to rats significantly enhanced the number of mesenchymal precursor cells in bone marrow with the ability to differentiate into an osteoblastic phenotype in ex vivo bone marrow cultures on day 4 of Exp 3. These studies provide evidence that short-term 1,25-(OH)2D3 treatment creates new bone remodeling units and augments osteoblast recruitment and osteoblast team performance in rat cancellous bone.

Effect of age and ovariectomy on fibroblastic colony-forming unit numbers in rat bone marrow.

It is now thought that osteoblasts (OB) stem from mesenchymal precursor cells (fibroblastic colony forming units or CFU-f) in the bone marrow. As the availability of these cells may have a profound effect on bone formation, the effect of age and ovariectomy (OVX) on CFU-f numbers was studied. It was found that with increasing age the numbers of CFU-f in the marrow were drastically reduced. OVX had no effect on CFU-f levels in 4 week and 4 month old rats but raised CFU-f levels in 1 year old rats back to levels seen in younger rats. SHam operation alone had an effect on CFU-f levels in older rats and the OVX effects were indistinguishable from the sham effect until 20 d post operation when the sham levels returned to baseline. The results show that CFU-f levels follow a similar trend to the rates of bone formation seen in aged and OVX rats.

Measurement of bone resorption by strontium excretion in prelabelled rats.

The body handles strontium (Sr) in a similar way to calcium (Ca) in that Sr is absorbed by the gut, concentrated in bone and excreted in urine and feces. In this study, rats were labelled with Sr during growth and later subjected to various treatments affecting bone resorption and Sr excretion was measured during and after treatment. Six weeks old Wistar rats were repeatedly s.c. injected with SrCl2. After a period of 2 weeks after the last Sr injection the rats were subjected to various treatments. Sr clearance was then measured weekly for 2 weeks. In the first experiment, the Sr labelled rats were sham-operated (sham) or ovariectomized (ovx) and urine collected afterwards. Sham rats were either treated with 4 daily s.c. clodronat injections at the beginning of the urine sampling, fed a low Ca diet (0.08% Ca) during the second sampling week or injected with saline. Urinary Sr excretion was decreased in the clodronate group during the first sampling week and increased in the Ca depleted group during feeding the low Ca diet. Sr excretion by ovx rats was similar to the sham control. In the second experiment, the effect of high-dose treatment with 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) or clodronate on bone resorption induced by Ca depletion was assessed by Sr output in urine and feces. Sr labelled rats were fed a low Ca diet and daily injected with 24,25(OH)2D3 or clodronate for 14 consecutive days. Clodronate significantly decreased Sr output during both sampling weeks. Treatment with 24,25(OH)2D3 resulted in an increased Sr output indicating an increase in bone resorption.

Physicochemical parameters of x-ray contrast media.

RATIONALE AND OBJECTIVES: Osmolality, including "dynamic osmolality," which is observed during dilution in the plasma, viscosity, density, and partition coefficient of 11 commercially available contrast media and two new nonionic monomers were determined. METHODS: Osmolality was measured by vapor pressure osmometry, viscosity by determining flow in microcapillaries, and partition coefficient in n-octanol or n-butanol/water mixtures by inductively coupled plasma mass spectroscopy or x-ray fluorescence analysis of iodine concentrations. RESULTS: For the commercially available contrast media, the following statistically significant ranking of osmolality was obtained at 300 mg iodine/mL: iotrolan << ioxaglate < iopromide < iopamidol < ioversol = iohexol < iopentol << meglumine diatrizoate. The novel nonionic monomers, ZK 119095 and ZK 139129, had very low osmolalities, and ZK 139129 was isotonic to blood. The partition coefficient for the system n-octanol/water was lowest for the ionic compounds ioxaglate and diatrizoate followed by the nonionic dimer iotrolan. In n-butanol, iotrolan showed the lowest partition coefficient. CONCLUSION: "Dynamic osmolalities" of contrast media may differ from static values possibly because of the formation of "quasi-oligomers." Especially for ZK 139129, disaggregation occurred during dilution and the osmolality increased slightly. However, osmolality was lower than for any other monomer during the whole dilution process.

Effect of niacin supplementation on the concentration of niacin in rumen and duodenal digesta and in whole blood of sheep.

1. Niacin concentrations in rumen liquor, duodenal digesta and blood, and daily duodenal flow of niacin were measured in four wethers which were fed an oatstraw grain concentrate diet (with normal niacin content) or an oatstraw maize diet (with low niacin content). The effect of an intraruminal supplementation of 2 mmoles nicotinic acid (NIA) or nicotinamide (NAM) on concentrations of NIA and NAM in gastrointestinal contents and blood and on daily duodenal flow of NIA and NAM was studied. The animals were equipped with rumen fistula and duodenal re-entrant cannulae. 2. NIA concentration in strained rumen liquor was 384 and 264 nmol/ml and daily duodenal flow of niacin was 616 and 528 mumoles/d in animals fed normal or low niacin diet, respectively. Intraruminal administrations of 2 mmoles of NIA or NAM raised NIA concentration in strained rumen liquor for approximately 6 h. About 20% of the administered dose of NIA or NAM passed out of the rumen and resulted in a 41% increase in daily duodenal flow of niacin. The remaining 80% of the administered dose of NIA or NAM disappeared from rumen liquor without being washed out. 3. Supplemented NAM was rapidly hydrolyzed to NIA and ammonia when administered into the rumen. 4. NAM concentration in blood appeared to be unaffected by the dietary content of NIA or by niacin supplementation. 5. The NIA concentration in strained rumen liquor was positively related with niacin content of the diet. Increases in duodenal flow of niacin had no marked effect on concentration of NAM in blood. It appeared that feeding diets with different NIA content affected apparent niacin synthesis in the rumen.

Normal plasma calcium, phosphate, and parathyroid hormone levels during 1,25(OH)2D3 infusions in rats.

The changes in plasma calcium, phosphate, and parathyroid hormone (PTH) levels that accompany 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] administration to experimental animals represent major obstacles to determining the precise role that 1,25(OH)2D3 plays in cell function in vivo. These difficulties arise because calcium, phosphate, and PTH have major cellular effects independent of 1,25(OH)2D3. To circumvent this obstacle, we have developed an animal model in which plasma 1,25(OH)2D3 levels were raised from 20 +/- 3 to 96 +/- 19, 240 +/- 49, and 459 +/- 66 pg/ml in vitamin D-deficient rats without influencing plasma ionized calcium, total calcium, phosphate, or NH2-terminal immunoreactive PTH (irPTH) levels. The elevated 1,25(OH)2D3 levels were achieved by constant subcutaneous infusion of 1,25(OH)2D3 using osmotic minipumps. Progressive reduction in the calcium and phosphorus content of the diet as the 1,25(OH)2D3 infusion rate was increased prevented concomitant changes in plasma calcium, phosphate, and irPTH levels. This experimental model, in conjunction with our previously developed normocalcemic rat model of vitamin D deficiency, provides a powerful experimental tool for the investigation of 1,25(OH)2D3 effects in vivo in the absence of concomitant changes in other parameters of calcium homeostasis.

Deficiency of vitamin D metabolites directly stimulates renal 25-hydroxyvitamin D3-1-hydroxylase activity in rats.

Renal 25-hydroxyvitamin D3-1-hydroxylase (1-hydroxylase) enzyme activity in rats is known to be increased by parathyroid hormone (PTH), hypophosphatemia, and hypocalcemia. Thus, enzyme activity is markedly increased in vitamin D-deficient states, but whether this stimulation is a direct response to the vitamin D deficiency or only occurs following the associated changes in plasma calcium, phosphate, or PTH is unclear. We tested whether vitamin D deficiency per se influences 1-hydroxylase activity in renal cortical slices using a normocalcemic rat model of vitamin D deficiency. Weanling male rats were fed one of the following three diets: (A) 0.8% Ca, 0.5% P, 2.2 IU vitamin D3/g; or vitamin D-deficient diets containing, (B) 0.8% Ca, 0.5% P; and (C) 2.0% Ca, 1.25% P, 20% lactose. Vitamin D-deficient rats fed diet B were hypocalcemic with elevated PTH at both test periods, and 1-hydroxylase activity was increased more than 100-fold compared with rats fed diet A. Plasma calcium, phosphate, and PTH levels were the same in groups A and C, but 1-hydroxylase activity was also substantially elevated in group C versus group A rats (104- and 17-fold increases after 10 and 19 diet weeks, respectively). These data lead to the important conclusion that severe deficiency of vitamin D metabolites per se provides a strong and independent stimulus to renal 1-hydroxylase activity in rats, perhaps due to the absence of 1,25(OH)2D3-mediated enzyme inhibition.

Normocalcemia without hyperparathyroidism in vitamin D-deficient rats.

Despite numerous attempts, no reliable dietary regimen exists to achieve vitamin D deficiency (-D) in rats without attendant changes in plasma parathyroid hormone (PTH), Ca, or phosphate. This represents an important obstacle to proper investigations of the physiologic role(s) of vitamin D metabolites in the function of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] target tissues. This paper describes the successful development of such a diet, which uses a combination of high Ca content, properly controlled Ca/P ratio, and lactose. Normal weanling rats were fed diets containing A, 0.8% Ca, 0.5% P, +D3, or -D diets containing B, 0.8% Ca and 0.5% P; C, 2.0% Ca and 1.25% P; or D, 2.0% Ca, 1.25% P, and 20% lactose. After 6 diet weeks group D rats remained normocalcemic and normophosphatemic, but diet groups B and C became hypocalcemic (6.9 +/- 0.8 and 7.2 +/- 0.4 mg/dl, respectively). Thus high dietary Ca and P was incapable of maintaining normal plasma Ca levels in the absence of dietary lactose. The normocalcemia in group D was not maintained by elevated PTH secretion because N-terminal PTH levels were also normal (14 +/- 3 versus 20 +/- 5 pg/ml). In contrast, PTH levels were markedly elevated in hypocalcemic groups B and C (47 +/- 7 and 48 +/- 10 pg/ml, respectively). Plasma 25-OHD3 and 1,25-(OH)2D3 levels were reduced to less than 120 and less than 12 pg/ml, respectively, in all -D groups. Thus the high-Ca diet and the use of normal weanlings did not impede the development of vitamin D deficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma Ca influences vitamin D metabolite levels as rats develop vitamin D deficiency.

The hypocalcemia that accompanies vitamin D deficiency is a major obstacle to proper interpretation of the role(s) of vitamin D metabolites in Ca-sensitive tissues. This paper describes the development and complete characterization of a dietary regimen with which normocalcemia was maintained in rats throughout the development of vitamin D deficiency. Normal weanling rats were fed diets containing 0.8% Ca, 0.5% P, and vitamin D3 (group A), or vitamin D-deficient diets containing 0.8% Ca and 0.5% P (group B); 2.0% Ca and 1.25% P (group C); or 2.0% Ca, 1.25% P, and 20% lactose (group D) for 19 wk. Group D rats were normocalcemic and normophosphatemic with normal parathyroid hormone (PTH) levels throughout the study. In contrast, from 4-19 diet wk, groups B and C were hypocalcemic with elevated PTH. Initially, plasma 25-hydroxyvitamin D3 [25(OH)D3] levels decreased most rapidly, and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] levels decreased least rapidly in group B rats, such that plasma 25(OH)D3 levels were reduced to 200-300 pg/ml before a decrease in 1,25(OH)2D3 levels was observed. However, vitamin D metabolite levels were similar in groups B, C, and D from 4-19 wk. Duodenal active Ca transport mirrored changes in plasma 1,25(OH)2D3 levels and was abolished after 10 wk. The results also suggested that vitamin D may not be necessary for normal bone mineralization since tibia mineral content and plasma alkaline phosphatase levels were similar in normocalcemic groups A and D throughout the study.

Purification procedure for the determination of niacin vitamers in gastrointestinal contents and blood.

A solid phase extraction procedure for subsequent simultaneous HPLC analysis of free nicotinic acid (NIA) and nicotinamide (NAM) in dried or liquid rumen and gastrointestinal contents from sheep is described. Twenty-five mg dried samples were suspended in 1.0 ml of 0.1 mol/l Li2SO4 and centrifuged. The supernatants were passed through two 500 mg solid phase extraction columns (Bond Elut NH2 and Sep Pak C18), mounted in series, and eluted by a four step pH gradient procedure. Recoveries of the two vitameres were between 70 and 75%. Purification was satisfactory when the method was applied to different fractions of rumen content (e.g. to food residues, to a microbial preparation and to rumen fluid), to intestinal contents of sheep and to blood. In rumen fluid no free NIA and NAM was found. Food particles and rumen microbes contained no free NAM.

Evaluation of elution parameters in reversed phase HPLC for the determination of niacin vitamers and metabolites.

A procedure for separation of nicotinic acid (NIA), nicotinamide (NAM), nicotinuric acid (NUR) and N1-methyl-nicotinamide (NMN) from biological samples by isocratic HPLC is described. The effects a) of different buffer systems (citrate, acetate, propionate), b) of different pH values (between pH 2.4 and 3.35), c) of different ionic concentrations of the eluents (between 10 and 100 mM) and d) of different methanol concentrations added to the eluent on retention times of the four compounds were tested. Results were described by multiple regression equations which allow selection of suitable analytical conditions according to the specific composition of different biological materials. The procedure yielded satisfactory separation of NIA and NAM from various purified fractions of rumen and intestinal contents and blood.