Sanford C. Garner

Estrogens reduce bone loss in the ovariectomized, lactating rat model.

Estrogen treatment of ovariectomized, lactating rats improved retention of bone mineral mass by 15-25% compared to ovariectomized, lactating rats receiving vehicle only. On the second day postpartum all lactating rats were ovariectomized and were placed along with age-matched non-mated controls on a whole-wheat flour-based diet with 0.1% calcium and 0.4% phosphorus. On day 6 postpartum estrogen treatment was begun with either implantation of a slow-release 17 beta-estradiol pellet or with the first of daily subcutaneous injections in sesame oil (vehicle). Increasing doses of estrogen resulted in decreased food consumption and decreased litter weight gain, both well-known effects of estrogens. Ovariectomized, lactating rats implanted with a slow-release pellet containing 0.35 mg 17 beta-estradiol had mean serum estradiol levels of 113.5 pg/ml. At the end of 21 days of lactation, femurs of dams with placebo pellets showed loss of 54% of bone ash weight compared with the non-mated controls versus only 42% loss by rats receiving estradiol treatment. Rats were also injected with estradiol benzoate in a sesame oil vehicle at 3 dose levels of 1.6, 5, or 16 micrograms/day. Only the 5 and 16 micrograms/day doses significantly improved retention of bone mineral mass during lactation (+17% and +18%, respectively, vs vehicle-injected, lactating rats). Estrone administered by subcutaneous injection also improved retention of bone during lactation; however, injection of 50 micrograms/day of estrone was required to produce an equivalent bone retention compared to 5 micrograms/day of estradiol. Thus, treatment of ovariectomized, lactating rats with estrogens results in a significant reduction of the loss of bone mineral mass associated with lactation.

The ovariectomized, lactating rat as an experimental model for osteopenia: calcium metabolism and bone changes.

The ovariectomized, lactating rat (Sprague-Dawley) is proposed as an experimental model for the rapid development of osteopenia which may be used to test the effectiveness of bone-retentive drugs potentially useful in treating osteoporotic women. Rats were ovariectomized (OVX) on day 2 postpartum and were kept on a low-calcium diet (0.1%). Measurements of serum total calcium, ionic calcium, albumin and parathyroid hormone were conducted between days 4 and 21 of lactation. Serum total and ionic calcium and albumin were significantly lower and serum parathyroid hormone was significantly higher in all lactating rats at 16 days postpartum compared to nonlactating controls. Mean bone mass of the femurs of OVX lactating rats measured at day 21 was approximately 50% of that of non-lactating intact controls. The enhanced duodenal calcium absorption (in everted gut sacs) associated with lactation was not affected by OVX and neither was the average litter weight gain between 2 and 14 days of lactation. In conclusion, lactation coupled with a low-calcium diet resulted in marked osteopenia, depressed serum calcium (both total and ionic) and significantly elevated serum parathyroid hormone concentration. The rapid and extensive bone loss of this model makes it appropriate for the study of therapeutic agents designed to retain bone mass.

Thyroid and blood thyrocalcitonin concentrations and C-cell abundance in two strains of rats at different ages.

Summary We have shown an increase in TCT in the blood and thyroid gland with increased age in two different strains of rats. Fischer (F344) rats, 6 to 18-months old, have a higher level of circulating TCT (-1.5 ng/ml) than young 10-week-old Fischer rats (<0.2 ng/ml). Thyroid TCT concentrations in old Fischer rats also were significantly higher than in young Fischer rats, and this was associated with a significantly higher area ratio of C-cells to follicular cells in the older rats. In Holtzman rats a similar increase in blood and thyroid TCT with age was also observed. Comparing the two strains, both thyroid and serum TCT in old Fischer rats were significantly higher than in old Holtzman rats. The authors wish to thank Drs. Paul L. Munson and Philip F. Hirsch for valuable suggestions during the course of this study. The authors also thank Dr. H. Stanley Bennett and Mr. Yukio Tanaka of the Laboratories for Reproductive Biology at the University of North Carolina for advice, criticism, and permission to use their equipment for the morphological study. The fine technical assistance of Mr. Johnny F. Obie and Mrs. Deloris B. Alston also is appreciated.

Characterization of choline metabolism and secretion by human placental trophoblasts in culture.

Choline is an essential nutrient for fetal development and may be utilized to form phospholipids such as phosphatidylcholine and sphingomyelin; to synthesize the neurotransmitter, acetylcholine; and to donate methyl groups after being oxidized to betaine. Since the majority of choline required for fetal growth must be transported by the placenta from the maternal circulation, we examined the ability of isolated human trophoblasts to metabolize choline and to release choline and its metabolites into culture medium. Cytotrophoblasts were isolated from normal, full-term human placentas and incubated with [14C]choline for 3 h; the cells were washed to remove extracellular radiolabel, and the changes in intracellular and medium choline pools were followed for an additional 24 h. During the incubation, choline rapidly reached steady state intracellularly and label was incorporated into betaine, phosphocholine, cytidylyldiphosphocholine, phosphatidylcholine, glycerophosphocholine, lysophosphatidylcholine, and sphingomyelin. All labeled choline metabolites in cells, except glycerophosphocholine, decreased at 6 and 27 h of incubation (3 and 24 h, respectively, after labeled choline was removed), and labeled metabolites appeared in media. By 24 h after labeled choline was removed, the major labeled metabolites in the media were choline (82%), betaine (11%), and glycerophosphocholine (5%). Small amounts of phosphatidylcholine (1%), and lysophosphatidylcholine (1%) were found. Acetylcholine was a very minor choline metabolite in these cells. When placental cells were incubated for 66 h after isolation, they formed syncytiotrophoblasts, which incorporated labeled choline into metabolites in a similar pattern to cytotrophoblasts. These data indicate that isolated trophoblast cells can metabolize choline to form all of its major metabolites and that several metabolites are released to the medium in significant amounts. Thus, our data suggest that the major metabolite supplied to the fetus may be choline, but that betaine and glycerophosphocholine may also be vehicles for transfer of choline equivalents from mother to fetus.

Interrelationships of bone ash and whole bone properties in the lactating and parous rat

SummarySeveral investigators have studied the mechanical properties of long bones of rats and have found that bone strength may be associated with bone mineral content. In this study we examined further the interrelationships of whole bone properties and bone ash weight of the rat femur. Using a 3-point bending test, the bones were evaluated during lactation and after one to three pregnancy— lactation periods—experiments in which bone ash was significantly reduced. A relationship was established between the time of lactation and the ash weight, stiffness, strength, and toughness but not the ductility. Using all of the experimental data, a highly significant relationship was observed between ash weight and stiffness. A similar significant correlation was revealed between bone ash and strength or toughness, although drawing individual lines for each experiment rather than a common line for all the experiments appeared more appropriate. Given the inherent inaccuracies in the material property measurements of bone, the results suggest that routine measurements of whole bone properties provide an important and sensitive way to evaluate bone quality and that these properties correlate significantly with the bone ash weight.

Acute lead-induced increase in serum calcium in the rat without increased secretion of calcitonin.

Summary An acute marked increase in total serum calcium was observed after a single iv injection of lead. The response was prompt, serum calcium rising above 20 mg/dl 60-90 min after doses ≥ 15 mg/kg. The hypercalcemia was due entirely to a rise in the non-dialyzable calcium fraction. In contrast, the ultrafilterable fraction which includes ionic calcium was actually diminished. This explains the absence of an increase in serum calcitonin despite severe hypercalcemia. A slight rise in total plasma proteins also was observed after lead injection but the significance of this observation remains to be determined. These acute effects of lead on calcium homeostasis are apparently different from its chronic effects since we have found, in a separate study, that rats exposed to lead for 1 year developed hyperplasia of C-cells and an increase in the levels of calcitonin in both the blood and the thyroid glands.

Controversy over dietary phosphorus.

The perspective presented by Leonard Sax in his Commentary, “The Institute of Medicine’s ‘Dietary Reference Intake’ for Phosphorus: A Critical Perspective,” provides persuasive evidence that the Institute of Medicine (IOM) may have given less attention to a potentially important adverse effect of high phosphorus intake accompanied by low calcium consumption, primarily resulting from poor food choices, than it deserves [1]. The issues raised by the author point clearly to the need for further human research on the long-term influence of a highphosphorus, low-calcium dietary pattern on the stimulation of parathyroid hormone and the eventual reduction of bone mass and density. At the current state of our knowledge base, it seems inappropriate to denigrate the IOM committee for its cursory examination of the high phosphorus issue [2], but rather it seems wiser to argue persuasively with NIH and other granting agencies to provide the appropriate funding to settle this perplexing question. A few points need to be elaborated on that make the argument of the author plausible, but a few cautionary comments are also in order. First, several animal model studies using mice, rats and dogs and a couple of well-designed human investigations, though short-term, support the concept that a high-phosphorus, low-calcium diet with a ratio of approximately 4 P:1 Ca (or worse) elevates serum parathyroid concentrations in a chronic fashion [3–5]. In the animal models, bone losses were extensive, but in the human investigations the study designs were too brief to expect to see any adverse skeletal effects [6–7]. A few observational human reports, largely based on cross-sectional data, suggest that the high phosphorus content of the US diet may be responsible for fractures in pre-adolescents and adolescents [8–9]. Unfortunately, the designs of these two studies have many limitations. Secondly, the author comes down hard on the IOM committee regarding its uncritical acceptance of published reports in support of its position that a high-phosphorus, low-calcium diet is not harmful to the skeleton, yet he does the same thing in giving carte blanche to the findings of published reports that favor his contention that adverse effects do result from such a diet [1]. Sometimes our biases get in the way of our own logic! Finally, the author makes a most acceptable recommendation at the conclusion of his report that further experimental investigation is necessary on this issue to sort out the facts from the fictions. One approach would be a prospective controlled trial of a sufficient number of older adults with appropriate checks and safeguards in place so that the investigation could be terminated if any identified adverse effects of such a diet were scientifically confirmed at some time into the study, especially if bone measurements showed, say at six months, a statistically significant decline from baseline. A forthcoming publication on phosphorus makes essentially the same point on the need for better data, but it also suggests that we need to assess better the hidden use of phosphate salts in foods because of their widespread use by the foods industry and by cola-type beverage producers [10]. Because of this latter concern about the high use of phosphates in food/beverage applications, federal regulatory agencies also need to enter into this issue. What we all really want is the truth of the matter on phosphorus, however it comes out. In summary, the current attack on the IOM report should stimulate both debate and controversy that hopefully will lead to further research on this conundrum.

Ontogeny of hepatic sn-1,2-diacylglycerol content and protein kinase C activity in the neonatal rat: lack of concordance

Altered activity of protein kinase C has commonly been related to activator-induced changes in cellular sn-1,2-diacylglycerol (1,2-DAG) concentration. In neonatal liver 1,2-DAG can be synthesized by the developmentally expressed monoacylglycerol acyltransferase (MGAT) activity (EC 2.3.1.22). Rat liver homogenats were examined on selected days after birth to determine whether the high MGAT activity present in neonatal rat liver was associated with high 1,2-DAG concentrations and altered protein kinase C activity and location. Although MGAT specific activity peaked between days 5 and 12, 1.2-DAG concentrations declined 63% between days 1 and 10, and the activity and membrane location of protein kinase C activity remained unchanged. Liver triacylglycerol content changed little during this time period, but the phospholipid and ceramide content of liver increased about 60 and 100%, respectively. Thus, changes in cell membrane 1,2-DAG content may not always be associated with changes in protein kinase C activity because multiple factors (including 1,2-DAG, fatty acids, and sphingosine) modulate the activity of this enzyme. Glycerolipid synthesis is likely to be the primary fate of the 1,2-DAG synthesized by the monoacylglycerol pathway.