Dietary determinants of post-menopausal bone loss at the lumbar spine: a possible beneficial effect of iron.

INTRODUCTION: Previous studies suggesting different effects of diet on post-menopausal bone loss may have given conflicting results because they sometimes failed to exclude confounding conditions or used imprecise methodology. DESIGN: To identify dietary determinants of bone loss from the lumbar spine after menopause in women not taking hormone replacement who developed no evidence of spondylotic or sclerotic degenerative disease, forty-three women were followed with repeated (mean = 12) measurements of bone mineral density (BMD) at L2-4 for 11-14 years. Eleven developed evidence suggestive of degenerative disease and were excluded. Diet was assessed at the beginning of the study and 2.5 years later using 3-day and 7-day periods of weighed intakes. Nutrients estimated were: carbohydrate, fat, protein, fibre, calcium, magnesium, iron, phosphorus, copper, zinc and six vitamins. We tested the ability of diet to predict post-menopausal bone loss using stepwise regression. RESULTS: Each woman's BMD change was described by a single coefficient after log transformation of the BMD data. The best model for BMD loss including dietary factors alone had two significant determinants: daily energy or protein (p=0.0003) intake was adverse, while dietary iron (p=0.002) was predictive of bone maintenance, an effect that persisted if iron was expressed as a ratio to energy intake. Adding body mass index to the model increased the goodness of fit (R (2)adj rose from 0.33 to 0.42) without affecting the statistical significance of the dietary determinants. CONCLUSIONS: Diet may influence bone loss after menopause, with dietary iron (or an associated factor) possibly having a protective effect on bone at the spine.

Prevalence of osteoporotic bone mineral density at the hip in Britain differs substantially from the US over 50 years of age: implications for clinical densitometry.

The purpose of this study was to compare hip bone mineral density (BMD) recorded in seven population based cohorts in Britain with the third National Health and Nutrition Examination Survey (NHANES III) US population-based reference data, in order to assess geographic variation in the prevalence of osteoporosis. Men and women aged 50-80+ years were randomly recruited from population and health registers. Dual X-ray absorptiometry (DXA) equipment was used to measure BMD at the hip, with the femoral neck and the trochanter regions studied. Prevalences of osteopenia and osteoporosis were estimated in accordance with World Health Organisation diagnostic criteria for women. Young normal data, used to establish cut-off criteria, was from NHANES III. Both male and female British subjects over 50-years-old were found to have significantly higher mean BMD at the femoral neck and trochanter than their US counterparts. Decline in BMD with age in British men appeared slower than in US men. Between British centres there were also statistically significant differences in BMD values in both sexes. British age-adjusted prevalences of osteopenia in women averaged 20% less than those of NHANES III, whereas the prevalence of osteoporosis was substantially lower in British subjects of both sexes (55% in women, 68% in men). Thus, applying the US NHANES III data as the referent, osteoporosis of the proximal femur in Britain appears to be less common than in the US, due primarily to differences in the lower tails of the BMD distributions. Providing that the relationship between fracture rates and BMD is the same in Britain and the US, it would still be appropriate to apply the reference data in fracture risk assessment in the UK.

Treatment with parathyroid peptides and estrogen replacement for severe postmenopausal vertebral osteoporosis: prediction of long-term responses in spine and femur.

Fifteen women with severe vertebral osteoporosis were treated with daily parathyroid peptide (hPTH) plus hormone-replacement co-therapy (HRT) for 1 year. Eight other patients were randomized to HRT alone. Co-therapy with hPTH and HRT resulted in an impressive mean treatment response at the spine (dual-energy X-ray absorptiometry DXA) 15% above baseline; P < 0.015 compared with the HRT group) at 2 years, while at the proximal femur and radius there were smaller increases. hPTH co-therapy led to a significantly positive metabolic calcium balance at 1 year (by 2.13 mmol Ca/day, equivalent to a 5% annual increment in total body calcium; P = 0.015). The magnitude of the lumbar spine DXA response at 2 years depended statistically on the increase in bone formation rate, measured with 85Sr (r2 adjusted 0.48; P < 0.005) and patients with a large spine DXA response had larger calcium balance improvements (P < 0.03). Plasma osteocalcin changes tracked closely with increases in bone formation rate (r2 = 0.87). In seven patients treated throughout with HRT alone, and in eight hPTH-treated patients (three of whom switched to bisphosphonate therapy at year 4). DXA spine changes seen in years 3-5 were minimal, with no evidence of a statistically significant difference between groups. It is concluded that hPTH or comparable PTH receptor activators remain the most promising anabolic treatment for osteoporosis currently under clinical evaluation and a 6- or 12-month measurement of bone formation or a marker predicts the 2-5 year bone density outcome. Post-hPTH treatment, loss of bone appeared preventable with anti-resorptive therapy.

Determinants of the first decade of bone loss after menopause at spine, hip and radius.

This study documented bone loss at three different sites in the early postmenopausal period, and examined potential predictors. Forty-three women underwent repeated measurements of bone density at the lumbar spine, proximal femur and distal radius for up to 14 years. Individual rates of bone loss were calculated for the spine and hip; for radial trabecular bone, rates were calculated separately for two time periods, earlier and later after menopause. In the spine and radius, initially high rates of loss diminished with time after menopause. No positive correlations for bone loss were found between the three sites, suggesting that faster than average bone loss was specific to individual bones. High body mass index (BMI) was significantly protective against fast bone loss at the spine and radius; in the spine, each unit increase in BMI was associated with a approximately 5% reduction in the rate of bone loss. Of the other variables measured (maximum oxygen consumption, lean body mass, fat mass, mean psoas muscle area at the L3 level, hand grip strength as well as anthropometry) only bone densitometry was sufficiently predictive to help guidance on hormone replacement or other prophylactic therapy. The data suggest that the known relationship between excessive leanness and risk of osteoporosis and vertebral fracture after menopause might in part be due to fast post-menopausal bone loss. Because bulk of psoas muscle was associated with low spine loss rates, the data also support a role for applied muscular loading in local maintenance of bone density.

Effects of four common anticonvulsants on the high pressure nervous syndrome in the rat.

This study reports results from experiments designed to test common, clinically useful anti-convulsants for their effectiveness, if any, against the high pressure nervous syndrome (HPNS) in rats. Phenytoin, carbamazepine, phenobarbitone, or diazepam were administered orally to rats before compression. Endpoints used to assess the progression of the HPNS were T1, T3, and T5 (onset of, continuous, and severe tremor), myoclonus, and seizures. Of the four drugs tested, only phenobarbitone increased the onset pressure for tremor and seizures by: T1 33%; T3 11%; T5 14%; seizures 10%. Neither phenytoin, carbamazepine, nor diazepam had any significant effect on any of the endpoints studied. High dose chronic pretreatment with phenytoin also had no effect on the HPNS. These data suggest that conventional anticonvulsant treatment would be of limited value for HPNS in man, and the lack of effect also suggests that HPNS seizures are of an unusual type.

Brain nuclei and neurotransmitters involved in the regulation of the high pressure neurological syndrome in the rat.

The role of glutamatergic (NMDA), cholinergic and purinergic neurotransmission in the pedunculopontine nucleus, red nucleus, ventrolateral thalamic nucleus, entopeduncular nucleus, and the substantia nigra in the development of the high pressure neurological syndrome (HPNS) was investigated in the rat. Focal injection of D-2-amino-7-phosphonoheptanoate (D-APH, 5 nmol per side) into the red nucleus or the pedunculopontine nucleus was protective against HPNS-induced convulsions. Carbachol (10 nmol), injected into the red nucleus, did not influence the severity of the symptoms of HPNS. Injection of carbachol into the pedunculopontine nucleus, significantly lowered the threshold pressure for convulsions and increased the threshold pressure for tremor. 2-Chloroadenosine (5 nmol), injected into the red nucleus, produced a potent antitremorgenic effect and a similar but less pronounced effect when injected into the pedunculopontine nucleus. 2-Chloroadenosine, injected into the substantia nigra (12.5 nmol) or the ventrolateral thalamic nucleus (25 nmol), facilitated the development of tremor and, in the entopeduncular nucleus (25 nmol), facilitated the occurrence of convulsions. These results show the complexity of neurotransmitter interactions in different regions of the brain, under high pressure. They also indicate that the biochemical and anatomical substrates, involved in the convulsions produced by HPNS, differ substantially from those in other experimental models of epilepsy.

Effects of four drugs on 4-aminopyridine seizures: a comparison with their effects on HPNS.

The effects of two i.v. anesthetics, Saffan and methohexitone (MHX) and two N-methyl-D-aspartate receptor antagonists, MK-801 (Dizocilpine) and 2-aminophosphonoheptanoate (AP7), were tested for activity against the motor excitation (tremor, whole body jerks, and seizures) produced by the K+ channel blocker 4-aminopyridine (4-AP). Saffan increased the dose of 4-AP required for all three endpoints; MHX had no effect on tremor but reduced the 4-AP required to produce jerks and seizures. MK-801 also reduced the 4-AP dose required to produce jerking but did not affect tremor or seizures. In contrast, AP7 increased the amount of 4-AP required to produce all endpoints. The effects of these drugs on 4-AP-induced excitation are similar to their actions on hyperbaric excitation, reported by us previously, and suggest that blockade of K+ channels may contribute to the high pressure nervous syndrome.

The high pressure neurological syndrome in genetically epilepsy prone rats: protective effect of 2-amino-7-phosphono heptanoate.

Genetically epilepsy prone rats (GEPR) are hypersensitive to various epileptogenic treatments and undergo characteristic generalized seizures when exposed to potent acoustic stimulation. We have studied the sensitivity of GEPR to high atmospheric pressure. Threshold pressures for behavioral symptoms of the high pressure neurological syndrome (HPNS) were recorded in normal Sprague-Dawley (SD) and GEPR (which originate from the SD strain) of both sexes. The threshold pressure (TP) for tremor and for convulsion was significantly lower in GEPR than in SD rats. The protective action of the NMDA receptor antagonist D-2-amino-7-phosphono-heptanoate (D-APH) was tested on both strains of rats. D-APH, 90 mg/kg ip was more protective against tremor in SD than in GEPR. Female GEPR were not protected against tremor. Protection against clonic seizures was similar in both sexes of GEPR and female SD rats while SD males were not significantly protected. None of the animals treated with D-APH developed the tonic phase of seizures. Blockade of the NMDA receptor with D-APH brought the threshold for convulsions in GEPR to a similar pressure to that obtained in SD vehicle-injected controls. This findings suggests the involvement of the excitatory amino acid system in the hypersensitivity of GEPR to high atmospheric pressure.

Effect of exposure to high pressure on subsequent spatial learning and memory in rats.

The effects of high helium pressure on the subsequent acquisition of spatial memory were studied in male rats. Thirty-two rats were exposed to 65 ATA helium-oxygen pressure for 4.2 days, decompressed (total time in chamber 5 days), and then tested in an eight-arm radial maze. Thirty-two control rats were exposed in the chamber to 1 ATA air. Each rat had 20 sessions in the maze (2 sessions/day for 10 days), and the number of correct (visiting an arm not previously visited to obtain the reward pellet) and incorrect choices (visiting a previously visited arm) were recorded. Statistical analysis showed that the rats exposed to 65 ATA performed significantly better than 1-ATA controls during the first 8 of 20 sessions. This effect was most pronounced in sessions 5-8. Results for sessions 9-20 showed that the pressure-treated rats still made more correct choices but to an extent that did not always reach statistical significance. Possible explanations include the pressure-treated rats performing better because of hunger after a lower food consumption at pressure. Alternatively, pressure itself may enhance proposed mechanisms of spatial memory such as long-term potentiation.

Exposure to high pressure may produce the 5-HT behavioral syndrome in rats.

In addition to the motor events associated with high pressure neurologic syndrome (HPNS), we have observed behavioral changes that resemble the 5-hydroxytryptamine (5-HT) syndrome in free-moving rats exposed to pressures up to 70 ATA. These include a flat body posture, head weaving, reciprocal forepaw treading, and hyperlocomotion. Such changes occur when brain 5-HT levels are raised or when 5-HT receptors are activated. We have therefore studied the behavior of rats at pressure treated either with saline or with one of the following drugs: p-chlorophenylalanine (pCPA) which depletes brain 5-HT by 85-90%, Wy 27587 which inhibits 5-HT reuptake, 5-hydroxytryptophan (5-HTP) and carbidopa which increase brain 5-HT synthesis, and quipazine which is a 5-HT receptor-agonist. After treatment, rats were individually exposed to pressure, and behavioral scores were made for 5 min every 10 ATA up to 70 ATA by an unbiased observer who was not aware of the treatment given. Analysis of all control rats indicated that only a flat body posture, forepaw treading, and hyperlocomotion were positively correlated with pressure, and these events were used in all subsequent analysis. Rats treated with pCPA with whole brain 5-HT levels reduced by 90% had scores significantly less than controls. Rats treated with Wy 27587 showed significantly increased scores. Rats treated with 5-HTP and quipazine failed to show a significant increase in scores. These results suggest that a modified form of the 5-HT syndrome occurs when rats are exposed to increased pressure, and the behavioral events seen are consistent with some activation of the 5-HT1A receptor subtype.

Effect of NMDA and 2-amino-7-phosphonoheptanoate focal injection into the ventrolateral thalamic nucleus on the high pressure neurological syndrome in the rat.

We report the effect of focal injections of N-methyl-D-aspartate (NMDA, 5 nmol) and 2-amino-7-phosphonoheptanoate (APH, 5 and 10 nmol) into the ventrolateral thalamic nucleus on behavioural symptoms of the high pressure neurological syndrome in rats. The injection of NMDA significantly lowers the threshold pressure for tremor and increases its intensity. The injection of APH significantly increases the threshold pressure for tremor and decreases its intensity. APH, 10 nmol, significantly increases the threshold pressure for myoclonus and convulsions. These protective effects are, however, less pronounced than those produced by either systemic injection of APH or its focal infusion into the basal ganglia output system.

The effects of non-competitive NMDA receptor antagonists on rats exposed to hyperbaric pressure.

The high pressure neurological syndrome (HPNS) occurs when man or animals are exposed to hyperbaric pressure. Four non-competitive N-methyl-D-aspartate (NMDA) antagonists - MK-801, phencyclidine (PCP), SKF 10,047 and ketamine were tested in rats for effects on the HPNS. All drugs were injected i.p. prior to compression; ketamine was also infused i.v. Control rats received saline. Rats were exposed individually to increasing helium pressure (PO2 0.5 atmospheres absolute ATA). Three endpoints were used to assess HPNS: onset pressures for tremor, myoclonus and convulsions. Neither MK-801 (0.03 and 0.3 mg/kg) nor SKF 10,047 (50 mg/kg) had any effect on the onset pressures for tremor, myoclonus or convulsions, although the type of seizure was modified from the clonic/tonic seizure seen in controls to purely clonic. PCP (5 mg/kg) had no effect on the endpoints, but pressure enhanced the excitation and stereotypy seen at 1 ATA. Ketamine (100 mg/kg i.p.) did not affect tremor or myoclonus; ketamine infused i.v. at pressure only prevented tremor and myoclonus at 'anaesthetizing' concentrations. Our results show that these non-competitive NMDA antagonists had little effect on HPNS, in contrast to competitive NMDA antagonists, such as AP7, which are highly effective. Possible explanations for this lack of effect include (1) interactions with NMDA receptor channels are pressure dependent; (2) other actions of these antagonists override their effects on the NMDA receptor channel.

Studies on the role of the NMDA receptor in the substantia nigra pars reticulata and entopeduncular nucleus in the development of the high pressure neurological syndrome in rats.

The effect of the focal injection of N-methyl-D-aspartate (NMDA) and 2-amino-7-phosphonoheptanoate (APH) into the substantia nigra pars reticulata (SNR) and entopeduncular nucleus (EP) on behavioural signs of the high pressure neurological syndrome (HPNS) in rats was studied. Doses of 1, 5 and 10 nmoles of NMDA or APH were injected into the SNR or EP, 10-30 min prior to the exposure of animals to a high pressure. Injection of NMDA into either SNR or EP results in a lowering of the threshold pressure for tremor by about 30%. Injection of NMDA into the SNR has no significant effect on clonic seizures whereas its injection into the EP results in a decrease of threshold pressure for clonic seizures. NMDA also facilitates the occurrence of forelimb clonus when injected into the EP. Injection of the NMDA antagonist, APH, into the SNR or EP significantly increases the threshold pressure of tremor (32.8 and 48.2% respectively). Seizure threshold is also increased by the injection of APH into either area, but nigral injections (especially the higher doses) are more protective against seizures than the EP injections. Comparing the two sites blockade of NMDA receptors within the EP is more protective against tremor, whereas in the SNR NMDA blockade is more protective against seizures.

The effects of kynurenic acid, quinolinic acid and other metabolites of tryptophan on the development of the high pressure neurological syndrome in the rat.

The effects of some biologically active metabolites of tryptophan on the high pressure neurological syndrome (HPNS) were studied. Kynurenic acid, quinolinic acid, 5-hydroxytryptophan, kynurenine and 3-hydroxyanthranilic acid, at doses within the physiological range, were administered exogenously to rats prior to exposure to increased pressure and any effects on the tremor, myoclonus and convulsion end points of the high pressure neurological syndrome were observed. Quinolinic acid (25 and 50 mg/kg) and kynurenine (50 mg/kg) reduced the onset pressure for tremor, but not myoclonus or convulsions. Kynurenic acid (100 mg/kg) increased tremor onset pressure; 5-hydroxytryptophan (20 mg/kg) slightly increased onset pressure for tremor but decreased that for myoclonus. 3-Hydroxyanthranilic acid (20 mg/kg) had no significant effect on any of the motor signs of the syndrome. These data provide further support for the idea that the motor events seen in the high pressure neurological syndrome are not produced by a single mechanism. Differences between the responses to related metabolites suggest that the precise balance between compounds such as kynurenic acid and quinolinic acid may be important in the appearance of the high pressure neurological syndrome.

Systemically administered glycine protects against strychnine convulsions, but not the behavioural effects of high pressure, in mice.

1. The effects of intraperitoneal administration of glycine were studied on the behavioural effects of raised ambient pressure in mice, compared with the effects of such administration on the actions of chemical convulsants. 2. Glycine did not alter the onset pressures for the occurrence of tremor, myoclonic jerks or clonic convulsions, when the ambient pressure was raised using helium. 3. Glycine showed a protective action against the convulsant effects of strychnine. 4. No protective action of glycine was found against the convulsant actions of pentylenetetrazol or bicuculline. 5. It is suggested that the results provide evidence that the high pressure neurological syndrome and strychnine convulsions have different neurophysiological origins.

Lack of correlation between the anaesthetic and anti-convulsant potencies of althesin, ketamine and methohexitone.

Using Sprague-Dawley rats, the anti-convulsant potencies of Althesin, ketamine and methohexitone were determined for bicuculline-and strychnine-induced seizures and compared with their effects on hyperbaric seizures. All three anaesthetics protected against both types of chemical convulsants; the degree of protection varied from 34 to 151%, with Althesin being the most effective. However, there was no correlation between their anti-convulsant and anaesthetic potencies, and no relationship between the effects on chemical convulsions and the interactions of the same agents with hyperbaric convulsions. These data suggest that the order of anti-convulsant potencies at equivalent anaesthetic concentration is Althesin much greater than ketamine = methohexitone, and that neither bicuculline-nor strychnine-induced seizures are a good model for hyperbaric convulsions.

The effect of two novel dipeptide antagonists of excitatory amino acid neurotransmission on the high pressure neurological syndrome in the rat.

Intracerebroventricular injection in the rat of beta-D-aspartyl aminomethylphosphonate (Asp-Amp) 1 mumol, or Y-D-glutamylaminomethylsulphonate (GAMS) 1 mumol, increases the onset pressure for the initial tremor phase of the high pressure neurological syndrome (HPNS) by 50%. Asp-Amp also significantly increases the onset pressures for myoclonus and for tonic-clonic seizures. GAMS did not significantly change the onset pressures for myoclonus or tonic clonic seizures, but it caused the appearance of brief clonic seizures prior to the onset of the HPNS.

Effect of exposing mice to 50 ATA helium pressure at different stages of pregnancy.

Pregnant T-O mice were exposed to 50 ATA He-O2 pressure for 4 days at different stages of gestation: 4-7, 6-9, and 9-12 days gestation. Controls were exposed to 1 atmosphere absolute (ATA) air. After the exposure period, pregnancy continued until 18 days gestation when the mice were killed and autopsied. Data were collected relating to the litters and placentas (Litter size, percent resorptions, placental weight, fetal-to-placental ratio) and fetuses (weight, crown-rump length, sex, skeletal abnormalities) and analyzed using analysis of variance. Results showed a small but significant increase in the percent resorptions in the pressure group and also a decrease in crown-rump length and placental weight. None of these changes were related to the stage of gestation in which the mice were exposed. No teratogenic effects of pressure were seen. We conclude that exposure to 50 ATA He-O2 during pregnancy in mice produces a small nonselective effect on fetal growth and development but does not affect any specific event taking place during these stages of embryogenesis.

Effect of barbituric acid on the high pressure nervous syndrome in the rat.

Male Sprague-Dawley rats were compressed in helium-oxygen mixture at a rate of 3 bar/min. Compression ended once the first convulsion was seen. Saline or barbituric acid at 1 or 2% were infused continuously during compression at a rate of 9.49 ml/h (tail vein). With barbituric acid at 1%, coarse tremor and convulsions appeared at higher pressures than in saline controls. No significant change was observed with 2% barbituric acid compared to saline controls. Barbituric acid which is nonanesthetic had some anti-high pressure nervous syndrome (HPNS) activity which is dose dependent. These results suggest that anti-HPNS activity of these different compounds involves at least one site which is different from that responsible for anesthetic activity.

Sperm maturation associated with subfertility following hyperbaric exposure of mice.

Mice exposed to 50 ATA heliox pressure during one spermatogenic cycle subsequently exhibit subfertility which can recover over an 8-wk period. This study was designed to investigate the changes in spermatid and testicular histology associated with these phenomena. Electron microscopy of testicular tissue from hyperbaric-exposed animals revealed a significant effect on spermatogenesis including disorganization and patchy necrosis of the epithelium. Two weeks after exposure to pressure, abnormalities were more difficult to find and tissues taken from animals 8 wk after exposure were indistinguishable from normal. These data directly confirm our earlier hypothesis that chronic hyperbaric exposure has a deleterious effect on sperm maturation, which is subsequently manifest as a failure in fertilizing capacity.