Evaluating and Strengthening the Evidence for Nutritional Bone Research: Ready to Break New Ground?

A healthy diet is essential to attain genetically determined peak bone mass and maintain optimal skeletal health across the adult lifespan. Despite the importance of nutrition for bone health, many of the nutritional requirements of the skeleton across the lifespan remain underexplored, poorly understood, or controversial. With increasingly aging populations, combined with rapidly changing diets and lifestyles globally, one anticipates large increases in the prevalence of osteoporosis and incidence of osteoporotic fractures. Robust, transparent, and reproducible nutrition research is a cornerstone for developing reliable public health recommendations to prevent osteoporosis and osteoporotic fractures. However, nutrition research is often criticized or ignored by healthcare professionals due to the overemphasis of weak science, conflicting, confusing or implausible findings, industry interests, common misconceptions, and strong opinions. Conversely, spurious research findings are often overemphasized or misconstrued by the media or prominent figures especially via social media, potentially leading to confusion and a lack of trust by the general public. Recently, reforms of the broader discipline of nutrition science have been suggested and promoted, leading to new tools and recommendations to attempt to address these issues. In this perspective, we provide a brief overview of what has been achieved in the field on nutrition and bone health, focusing on osteoporosis and osteoporotic fractures. We discuss what we view as some of the challenges, including inherent difficulties in assessing diet and its change, disentangling complex interactions between dietary components and between diet and other factors, selection of bone-related outcomes for nutrition studies, obtaining evidence with more unbiased designs, and perhaps most importantly, ensuring the trust of the public and healthcare professionals. This perspective also provides specific recommendations and highlights new developments and future opportunities for scientists studying nutrition and bone health. © 2021 American Society for Bone and Mineral Research (ASBMR).     

The response to ether anesthesia and to electric stimulation of glycolytic metabolites in a red and a white muscle of the rat

Summary Since ether anesthesia lowered ATP by 25% in red, but not in white muscle, and only when the spinal neurones were intact, we suggested that small or intermediate muscle units were activated under ether anesthesia [8].In order to prove this postulate, some glycolytic metabolites, known to rise under muscular activation, are studied in the white musculus adductor magnus and in the red musculus pyramidalis of the rat: glucose-1-phosphate, glucose-6-phosphate, fructose-6-phosphate, fructose-1-6-diphosphate, -glycerophosphate, lactate, pyruvate, and dihydroxyacetone phosphate.The conditions compared are: Inactin (5-ethyl-5(methyl-propyl)-2-thiobarbituric acid)-anesthesia, diethyl ether anesthesia, and tetanic contraction under Inactin anesthesia.The histological assay with Sudan-black B staining shows 34.2±7.3% dark fibers in m. pyramidalis and 0.2±4.8% dark fibers in m. adductor magnus.Glucose-1-phosphate, fructose-1-6-diphosphate, and -glycerophosphate are elevated under ether anesthesia in both muscles versus Inactin anesthesia by 100–200%.Lactate in both muscles and pyruvate in the red muscle are slightly elevated under ether (by 40%) versus Inactin anesthesia.Under tetanic contraction the metabolites studied rise considerably in both muscles.As glycogen is lowered in rat muscle under ether [9], the present results suggest an activation of glycogen phosphorylase and of phosphofructokinase in both the red and the white muscle under ether anesthesia, which results in augmented glycolysis.The comparatively small increment of pyruvate and lactate in the presence of a high increment of -glycerophosphate under ether anesthesia is considered to indicate an asynchronous activation of fibers with unimpaired circulation and oxidative metabolism.     

Golgi-like demonstration of “dark” neurons with an argyrophil III method for experimental neuropathology

Summary A silver method is proposed for the selective, well-contrasted and reproducible demonstration of dark neurons in frozen, vibratome and paraffin sections cut at a thickness of 5 to 200 m from aldehyde-fixed brains. The Golgi-like staining of the dendrites enables asorting of dark neurons according to characteristic neuron classifications. The staining procedure includes an esterification with 1-propanol, a treatment with diluted acetic acid and development. The esterification strongly increases the argyrophilia of both dark neurons and mitochondria. Unwanted co-staining of mitochondria is suppressed by the acetic acid treatment, while a special developer is used to render the staining controllable. The applicability of the method to experimental neuropathology is demonstrated by Golgi-like staining of dark neurons in rat brains exposed, before transcardial perfusion-fixation and delayed autopsy, to various pathological conditions including ischemia, hypoglycemia, trauma, status epilepticus, deafferentation and poisoning with kainic acid, colchicine and sodium azide, respectively.     

Epothilone A induces apoptosis in neuroblastoma cells with multiple mechanisms of drug resistance

Epothilone A, a novel macrolide antibiotic, is produced by the myxobacterium Sorangium cellulosum. Similarly to paclitaxel (Taxol), epothilone A inhibits cell proliferation and induces apoptosis by binding to tubulin and stabilizing of microtubuli. Like paclitaxel, epothilone A induced apoptosis in neuroblastoma cells which exhibit constitutive cytoplasmic sequestration of p53 and, hence, an impaired DNA-damage-dependent apoptosis. However, in contrast to paclitaxel, epothilone A was also effective against a constitutively Pgp-expressing, multidrug resistant neuroblastoma cell line (SK-N-SH). Moreover, the efficacy of epothilone A was not impaired even though the Pgp level was further increased during treatment with the drug.     

Investigations with the selective PKC inhibitor chelerythrine on human basophils

Modulation of protein kinase C (PKC) activity in basophils (B) can influence IgE-mediated histamine release (HR). The present study investigated the effects of chelerythrine, which inhibits isolated PKC (IC₅₀ 0.7 M), on different activation pathways in B. FcRI-mediated HR was strongly inhibited by chelerythrine (86.5±5.4%, 5 M,n=11). TPA-induced mediator release was also suppressed: 77.1±8.5% inhibition (7.5 M). HR due to non-immunological stimuli (A23187, FMLP) was strongly inhibited by chelerythrine. Previously, other selective PKC-inhibitors have been shown to potentiate IgE-mediated HR from B suggesting a negative modulatory role of PKC, whereas non-specific inhibitors such as staurosporine inhibited HR. Chelerythrine might therefore be less selective for PKC. This may be indicated by the fact that chelerythrine inhibits PKC at its catalytic domain, which is homologous with other protein kinases.     

Incidental cholecystectomy during colorectal surgery.

OBJECTIVE: To assess the risks and benefits of incidental cholecystectomy in patients having colorectal surgery. SUMMARY BACKGROUND DATA: Cholelithiasis is found commonly during abdominal surgery. Previous studies used disparate methods to assess the risks and benefits of incidental cholecystectomy and have reached contradictory conclusions. METHODS: All patients in whom asymptomatic cholelithiasis was noted during colorectal surgery between January 1982 and December 1986 were studied. Operative morbidity and long-term outcome were assessed by chart review and questionnaire. RESULTS: Three hundred five patients were identified, of whom 195 (63.9%) had an incidental cholecystectomy and 110 (36.1%) did not. The two groups were similar in terms of age, sex, primary disease, and associated medical conditions, although fewer emergency procedures, abdominoperineal resections, and Hartmann's procedures were needed in the cholecystectomy group. The overall operative morbidity rate was the same in both groups. The long-term risk for developing small bowel obstruction was also similar. After a median follow-up of 6 years after hospital discharge, biliary pain or cholecystitis developed in 16 patients (14.6%) in the "no cholecystectomy" group, 12 of whom have had cholecystectomy. Two additional patients had cholecystectomy for acute postoperative cholecystitis while still in the hospital. Six more patients have had incidental cholecystectomy at subsequent laparotomies. The cumulative probability of needing cholecystectomy at 2 and 5 years after the initial colorectal operation was 12.1% and 21.6%, respectively. CONCLUSIONS: Incidental cholecystectomy was not associated with increased postoperative morbidity, whereas the long-term risk that previously asymptomatic gallstones would become symptomatic was substantial. Unless there are clear contraindications, patients with asymptomatic gallstones who have colorectal surgery should have concomitant cholecystectomy.     

Calcitriol oral pulse therapy in children with renal osteodystrophy

A 6-month protocol of oral pulse calcitriol was used in nine uraemic children (2–14 years old) on dialysis who presented with renal osteodystrophy. Calcitriol was administered twice a week, 4 g per dose for patients over 30 kg and 3g for patients less than 30 kg. Plasma levels of parathyroid hormone, calcium, phosphorus and alkaline phosphatase were carefully controlled during the study. Parathyroid hormone levels decreased by 68% and 56% by the 2nd and 6th months of treatment in seven patients, while they remained unchanged in two patients with focal segmental glomerulosclerosis and massive proteinuria. Eight hypercalcaemic episodes from 77 determinations were observed, all of them recovered after 1 week of vitamin D withdrawal. We conclude that oral calcitriol pulse therapy is a good alternative for renal osteodystrophy in uraemic children. Careful monitoring of plasma parathyroid hormone and calcium is needed during follow-up when using this approach in paediatric patients.     

Using qualitative methods to elicit recall of a critical time period

The period during and after puberty seems to be important for breast cancer initiation. Because experiences during that time are likely to be influenced by a woman's cultural background, we conducted a pilot study among Hispanic and Caucasian women to elicit their memories of early life events. These data were used to design culture-specific questionnaire modules for the retrospective assessment of peripubertal breast cancer risk factors, using specific strategies to trigger accurate recall. Study subjects were volunteer breast cancer survivors or relatives of survivors. In carrying out this work, we took methods from the social sciences and applied them to a research question in chronic disease epidemiology. We found both qualitative and quantitative differences in recall of peripubertal exposures and experiences between Hispanic and Caucasian subjects. Our preliminary data indicate that in contrast to Caucasian women, Hispanic women consider the church rather than school a touchstone for recalling past events. Under the domain "body development," Hispanic women are more likely to mention menstruation than Caucasian women but less likely to recall changes in body hair and breast development. Caucasian women cited team sports as an important physical activity during the peripubertal period, whereas Hispanic women listed more sedentary games and housework as the main activities. Results of our pilot study support the view that to enhance the validity of retrospective data on peripubertal breast cancer risk factors, it is important to take account of cultural differences. Our experience using qualitative methods to elicit data of this kind in the context of a larger epidemiologic research effort suggests that such innovative approaches are valuable.     

Estrogenic potential of certain pyrethroid compounds in the MCF-7 human breast carcinoma cell line

Estrogens, whether natural or synthetic, clearly influence reproductive development, senescence, and carcinogenesis. Pyrethroid insecticides are now the most widely used agents for indoor pest control, providing potential for human exposure. Using the MCF-7 human breast carcinoma cell line, we studied the estrogenic potential of several synthetic pyrethroid compounds in vitro using pS2 mRNA levels as the end point. We tested sumithrin, fenvalerate, d-trans allethrin, and permethrin. Nanomolar concentrations of either sumithrin or fenvalerate were sufficient to increase pS2 expression slightly above basal levels. At micromolar concentrations, these two pyrethroid compounds induced pS2 expression to levels comparable to those elicited by 10 nM 17ss-estradiol (fivefold). The estrogenic activity of sumithrin was abolished with co-treatment with an antiestrogen (ICI 164,384), whereas estrogenic activity of fenvalerate was not significantly diminished with antiestrogen co-treatment. In addition, both sumithrin and fenvalerate were able to induce cell proliferation of MCF-7 cells in a dose-response fashion. Neither permethrin nor d-trans allethrin affected pS2 expression. Permethrin had a noticeable effect on cell proliferation at 100 microM, whereas d-trans allethrin slightly induced MCF-7 cell proliferation at 10 microM, but was toxic at higher concentrations. Overall, our studies imply that each pyrethroid compound is unique in its ability to influence several cellular pathways. These findings suggest that pyrethroids should be considered to be hormone disruptors, and their potential to affect endocrine function in humans and wildlife should be investigated.