Undercarboxylated osteocalcin and bone mass in 8-12 year old children with cystic fibrosis.

UNLABELLED: Young adults with cystic fibrosis (CF) frequently develop bone disease. One suggested aetiological factor is suboptimal vitamin K status with impaired carboxylation of osteocalcin and abnormal bone formation. METHODS: We measured bone mineralization and turnover in thirty-two 8-12 year old CF patients (14 boys) using Dual Energy X-ray absorptiometry (whole body (WB) and lumbar spine (LS)), 25-OH Vitamin D, PTH and markers of bone formation (plasma osteocalcin, N-terminal pro-peptide of type 1 collagen (P1NP)), plus an indirect measure of vitamin K status, undercarboxylated osteocalcin (uc-OC). RESULTS: LS bone mineral density (BMD) standard deviation (SD) scores were < -1.0 in 20% of subjects. Size-adjusted LS and WB bone mass was normal. Compared to reference data, % uc-OC was high and P1NP low. LS bone mass was predicted by % uc-OC but not other markers (0.4% decrease in size-adjusted LSBMC (p=0.05); 0.04 SD decrease in LSBMAD (p=0.04) per 1% increase in uc-OC). CONCLUSION: Markers suggestive of sub-optimal vitamin K status and low bone formation were present despite normal size-adjusted bone mass. The association between LSBMC and % uc-OC is consistent with the hypothesis that sub-optimal vitamin K status is a risk factor for CF bone disease. This should ideally be investigated in an intervention trial.

Positive, site-specific associations between bone mineral status, fitness, and time spent at high-impact activities in 16- to 18-year-old boys.

The incidence of hip and forearm fracture in elderly men in the United Kingdom is a public health issue, but there is limited knowledge on lifestyle factors affecting male bone health. The aim of this cross-sectional study was to evaluate the relationships between whole body and regional bone mineral status and self-reported participation time in no-, low-, moderate-, and high-impact activities and fitness measurements in 16- to 18-year-old boys. One hundred twenty-eight boys underwent absorptiometry (DXA) measurements (Hologic QDR 1000W) of bone mineral content (BMC), bone area (BA), and bone mineral density (BMD) at the whole body, hip, spine, and forearm. They also completed the EPIC (European Prospective Investigation of Cancer) physical activity questionnaire, which allowed categorization of activities according to impact and aerobic intensity. Fitness and strength were assessed in each subject using estimated VO2 max, grip strength, and back strength. Significant positive relationships were found between BMC, BA, and BMD and the fitness and strength measurements and participation time in high-impact sports at most skeletal sites. The relationships were further examined after adjustment of BMC for height, weight, and bone area, thereby minimizing the influence of body and bone size on these relationships. VO2 max was a significant positive determinant of size-adjusted BMC at the whole body, the ultradistal and one-third radius, and all the hip sites, except the trochanter. Size-adjusted BMC at the forearm sites and trochanter was significantly positively associated with grip strength. Size-adjusted BMC at the whole body and all the hip sites was significantly positively associated with time spent at high-impact activities. Differences in size-adjusted BMC across thirds of time spent at high-impact activities were also examined. Boys in the highest third of high-impact activity had significantly greater size-adjusted whole body BMC and total hip BMC compared to subjects in the lowest third [+3.4 (1.2)% for whole body and +8.5 (2.2)% for hip, both P = 0.001]. Boys in the highest third of high-impact activity spent most activity time jogging, playing tennis, football and rugby, basketball, and exercising with weights. Back strength and lean mass were significantly greater in subjects in the highest third compared to those in the middle (P = 0.02) and lowest third (P = 0.03). No significant differences were found between subjects in each third of high-impact activity for potential confounders including TV viewing, calcium intake, body fat, and height. These findings indicate that participation of male adolescents in a range of high-impact activities for 1 h or more a day is associated with greater bone size and mineral content, especially at the hip.

Inter and intra-individual variations in urinary excretion of pyridinium crosslinks of collagen in healthy young adults.

OBJECTIVES: To investigate the inter- and intra-individual variation in the excretion of pyridinium crosslinks of collagen in first morning void urine samples in healthy young adults. DESIGN: An observational study in which urinary pyridinium crosslinks of collagen were measured in fasting first void urine samples over five consecutive days. SETTING: The study was conducted in the Department of Nutrition, University College, Cork, Ireland. SUBJECTS: A total of 17 healthy young adults (11 females and 6 males) aged 22-26 y were recruited from University College, Cork. The subjects were without any history of bone or articular disease, and with no intake of medicine that could affect bone or cartilage metabolism. RESULTS: There were large intra-individual variations in the excretion of both pyridinoline (Pyr) and deoxypyridinoline (Dpyr) in first morning void urine samples and the ratios of intra- to inter-individual variation were high. In comparison, inter-individual variation in excretion of crosslinks was relatively low. No significant day-to-day variation was observed either in Pyr or Dpyr excretion when either five, four or three consecutive days or four or three non-consecutive days of urine sampling were used. However, there were significant differences between any two consecutive or non-consecutive days in both Pyr and Dpyr excretion. CONCLUSIONS: The relatively large day-to-day variation in urinary excretion of the pyridinium crosslinks emphasises the need to use multiple samples to characterise the crosslink excretion of an individual. The present study demonstrates that urine sampling for these crosslinks necessitates at least a 3 d collection to ensure that day-to-day effects are not a significant source of variation included in the data analysis.

Effects of usual nutrient intake and vitamin D status on markers of bone turnover in Swiss adolescents.

OBJECTIVE: To evaluate the effects of nutrient intake and vitamin D status on markers of type I collagen formation and degradation in adolescent boys and girls. DESIGN: Cross-sectional study. SETTING: Canton of Vaud, West Switzerland. SUBJECTS: A total of 92 boys and 104 girls, aged 11-16 y. Data were collected on height, weight, pubertal status (self-assessment of Tanner stage), nutrient intake (3-day dietary record) and fasting serum concentration of 25-hydroxyvitamin D (25OHD), and markers of collagen formation (P1NP) and degradation (serum C-terminal telopeptides: S-CTX). RESULTS: Tanner stage was a significant determinant of P1NP in boys and girls and S-CTX in girls. Of the nutrients examined, only the ratio of calcium to phosphorus (Ca/P) was positively associated with P1NP in boys, after adjustment for pubertal status. 25OHD decreased significantly at each Tanner stage in boys. Overall, 15% of boys and 17% of girls were identified as being vitamin D insufficient (serum 25OHD <30 nmol/l), with the highest proportion of insufficiency at Tanner stage 4-5 (29%) in boys and at Tanner stage 3 (24%) in girls. A significant association was not found between 25OHD and either bone turnover marker, nor was 25OHD insufficiency associated with higher concentrations of the bone turnover markers. CONCLUSIONS: The marked effects of puberty on bone metabolism may have obscured any possible effects of diet and vitamin D status on markers of bone metabolism. The mechanistic basis for the positive association between dietary Ca/P ratio and P1NP in boys is not clear and may be attributable to a higher Ca intake per se, a critical balance between Ca and P intake or higher dairy product consumption. A higher incidence of vitamin D insufficiency in older adolescents may reflect a more sedentary lifestyle or increased utilisation of 25OHD, and suggests that further research is needed to define their requirements. SPONSORSHIP: Nestec Ltd and The Swiss Foundation for Research in Osteoporosis.

Dietary acid-base balance and intake of bone-related nutrients in Cambridge teenagers.

OBJECTIVES: To evaluate the diet of 16-18-y-old boys and girls with particular reference to intakes of nutrients believed to affect bone health and dietary acid-base balance. DESIGN: A 7-day food diary was completed between the months of October and December. SETTING: Cambridge, UK. SUBJECTS: A total of 111 boys and 101 girls aged 16-18 y who were recruited into the Cambridge Bone Studies. MAIN OUTCOME MEASURES: Mean daily intakes of foods and selected nutrients (protein, calcium, phosphorus, magnesium, potassium, vitamins C and K) were calculated. Two estimates of acid-base balance were calculated from the diet using the formulae of Remer (net acid excretion, estimated indirectly; NAE(ind)) and Frassetto (protein/potassium ratio). RESULTS: Mean calcium and phosphorus intakes were above the UK Reference Nutrient intake (RNI). In all, 39% of the boys and 36% of the girls had vitamin K intakes lower than 1 microg/kg body weight/day. Calcium intake was positively correlated with all other nutrients except vitamins C and K. Boys had a significantly higher estimated net acid excretion (NAE(ind)) than girls (P<0.001). Although a strong correlation (r=0.76, P<0.001) was found between the two methods, at higher acid levels a divergence was observed. A significant positive correlation was found between NAE(ind) and the weight consumed per day of milk, cheese, meat and cereal foods and a negative correlation was found with the weight of potatoes and fruit. Diet composition is such that a lower NAE(ind) is accompanied by a lower calcium intake. CONCLUSIONS: The interpretation of the effects of calcium and other nutrients on bone cannot be considered in isolation from the other components of the diet. These results challenge some of the accepted perceptions about what constitutes an optimal diet for the promotion of bone health in adolescents.

No effect of copper supplementation on biochemical markers of bone metabolism in healthy young adult females despite apparently improved copper status.

OBJECTIVE: To investigate the effects of increasing Cu intakes, above the usual dietary intake, on biomarkers of bone metabolism in healthy young adult females (aged 21-28 y) over a 4 week period. DESIGN: A double-blind, placebo-controlled randomised repeat crossover Cu supplementation trial. SETTING: The study was conducted at the Royal Veterinary and Agricultural University (RVAU), Copenhagen, Denmark. SUBJECTS: Sixteen healthy young adult females aged 20-28 y were recruited from among students at the RVAU. INTERVENTION: During the 4 week intervention periods in this randomised, crossover trial (3x4 weeks with a minimum 3 week wash-out period), each subject received, in addition to their usual diet, either 3 or 6 mg elemental Cu/day as CuSO4 or a matching placebo. On the last 3 days of each dietary period 24 h urines were collected. In addition, blood was collected on the last day of each dietary period. RESULTS: Serum Cu and erythrocyte superoxide dismutase (but not caeruloplasmin protein concentration or activity (putative indices of Cu status)) were significantly increased (P<0.05) after daily Cu supplementation with 3 and 6 mg/day for 4 weeks. Serum osteocalcin (biomarker of bone formation), urinary creatinine (Cr) concentration, urinary pyridinoline (Pyr)/Cr or deoxypyridinoline (Dpyr)/Cr excretion, or daily urinary Pyr or Dpyr excretion (biomarkers of bone resorption) were unaffected by Cu supplementation. CONCLUSION: Copper supplementation of the usual diet in healthy young adult females, while apparently improving Cu status, had no effect on biochemical markers of bone formation or bone resorption over 4 week periods. SPONSORSHIP: Funding from the European Commission.

Interrelation of parathyroid hormone and vitamin D metabolites in adolescents from the UK and The Gambia.

Parathyroid hormone (PTH) is used as a marker of vitamin D (VD) status. However, PTH depends on many other factors. The 24,25-dihydroxy VD (24,25VD) concentration may be a sensitive marker because its production is reduced in VD deficiency. The relationship between VD metabolites, their ratio and PTH was investigated in adolescents from the UK and The Gambia with different calcium intakes and VD status. In the UK, there was a significant positive (+ve) association between 25VD and both 1,25-dihydroxy VD (1,25VD) and 24,25VD and a negative (-ve) association with PTH. The 24,25:25VD ratio was consistent across the 25VD concentration range. There was a +ve association between PTH and 1,25:25VD, (1,25+24,25):25VD or 1,25:24,25VD, a -ve association with 24,25VD and none with 1,25VD or 24,25:25VD. Using LnPTH and 1,25:25VD ratio (but not 1,25VD:24,25VD or 25VD:24,25VD) increased uniformity between groups and strength of relationships compared to PTH and 1,25 or 25VD alone. In The Gambia, there was a significant -ve relationship between 25VD and PTH and none with 1,25VD. There was a +ve association between 1,25VD or 1,25:25VD and PTH. The more uniform prediction of PTH by the 1,25VD:25VD ratio may be because this better reflects the extent to which PTH-induced 1,25VD production can be met by VD supply. Further validation is needed.

Assessment of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D3 concentrations in male and female multiple sclerosis patients and control volunteers.

Populations with insufficient ultraviolet exposure and who consume diets low in vitamin D have low vitamin D status (plasma 25-hydroxyvitamin D (25(OH)D) concentrations) and a reported higher incidence of multiple sclerosis (MS). The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is an effective anti-inflammatory molecule. No research to date has assessed 1,25(OH)2D3 concentrations in individuals with MS. In this study, plasma concentrations of 25(OH)D, 1,25(OH)2D3 and parathyroid hormone (PTH) were measured in 29 individuals with MS and 22 age- and sex-matched control volunteers. There were no significant differences in plasma PTH, 25(OH)D and 1,25(OH)2D3 concentrations between individuals with MS and control volunteers. Women with MS had significantly higher 25(OH)D and 1,25(OH)2D3 concentrations than men with MS (79.1+/-45.4 versus 50.2+/-15.3 nmol/L, P=0.019 and 103.8+/-36.8 versus 70.4+/-28.7 pmol/L, P=0.019, respectively). There was a significant positive correlation between 25(OH)D and 1,25(OH)2D3 concentrations in all subjects (r=0.564, P=0.000), but secondary analysis revealed that the correlation was driven by women with MS (r=0.677, P=0.001). Significant sex differences in vitamin D metabolism were observed and were most marked in individuals with MS, suggesting that vitamin D requirements may differ between the sexes, as well as by underlying disease state.

Relationships between biochemical bone turnover markers, season, and inflammatory status indices in prepubertal Gambian boys.

Seasonal and interindividual differences in food availability and illnesses cause variations in growth, including bone growth, in children in developing countries. We investigated seasonal differences in biochemical (serum) markers of bone metabolism and relationships between these markers (procollagen type I N propeptide [P1NP], serum C-terminal telopeptide of type 1 collagen [S-CTX]) and serum markers of inflammation (alpha(1)- antichymotrypsin [ACT], C-reactive protein [CRP], sialic acid) in prepubertal Gambian boys. Three seasonal time points were chosen: August, mid-rainy season; October, late rainy season (both are associated with decreased food supply, increased prevalence of infection, reduced weight gain, and stunting); and April, late dry season, when environmental conditions are better and rates of weight gain are higher. The prevalence of raised inflammatory markers was high: 29% of ACT values >0.40 g/L in August, 42% >0.40 g/L in October, and 18% >0.40 g/L in April. Of CRP values, 92-96% were >0.47 mg/L and 30%, 46%, and 18% were >3.95 mg/L in August, October, and April, respectively. Also, 42-52% of sialic acid values were >648 mg/L. Of the bone markers, S-CTX exhibited the highest values in August; P1NP did not vary with season. Inverse relationships between each bone marker and the acute phase markers occurred, with the strongest relationships between P1NP and ACT or sialic acid. We conclude that bone collagen synthesis and turnover are reduced during inflammation in rural Gambian boys. Biochemical indices can provide powerful, single-time point evidence of infection and growth-faltering episodes.

PIXImus bone densitometer and associated technical measurement issues of skeletal growth in the young rat.

The PIXImus dual-energy X-ray absorptiometer (DXA) is designed to measure body composition, bone mineral content (BMC), area (BA), and density (BMD) in mice and rats. The aims of this study were to longitudinally measure BMC, BA, and BMD in growing rats and to identify potential technical problems associated with the PIXImus. Total femur and lumbar DXA measurements, body weight, and length of initially 3-week-old rats (n = 10) were taken at weeks 5, 9, and 14. BMC and BMD of femoral metaphyseal and diaphyseal regions rich in trabecular and cortical bone, respectively, were obtained. Results showed significant increases in body weight, total femur BMC and BMD, lumbar area, length, BMC, and BMD at each time point. There was a significant positive correlation between body weight and total femur BMD (r = 0.97, P < 0.001) as well as lumbar BMD (r = 0.99, P < 0.001). BMD values for the femoral metaphyseal region and the lumbar spine were also positively correlated (r = 0.96, P < 0.01). Several technical issues (e.g., positioning of animals), difficulties (e.g., in analysis of images), and limitations (e.g., inability to detect underdeveloped calcified bone in growing animals and bone edge detection) of the software pertinent to the PIXImus were evident. In conclusion, despite limitations in the software, the PIXImus is a valuable tool for studying skeletal development of growing rats.

The effect of short-term calcium supplementation on biochemical markers of bone metabolism in healthy young adults.

The influence of Ca supplementation of the usual diet for 14 d on biochemical markers of bone turnover was investigated in healthy young adults aged 21-26 years. In a crossover study, eighteen subjects (five male and thirteen female) were randomly assigned to their self-selected diet (about 22 mmol Ca/d) or their self-selected diet with a 20 mmol/d Ca supplement (about 40 mmol Ca/d) for 14 d followed by crossover to the alternative diet for a further 14 d. During each dietary period fasting morning first void urine samples (last 3 d) and fasting blood serum samples (morning of twelfth day) were collected. Ca supplementation reduced urinary excretion of pyridinoline (14%) and deoxypyridinoline (16%) (biochemical markers of bone resorption) but had no effect on biochemical markers of bone formation (serum osteocalcin and bone-specific alkaline phosphatase; EC 3.1.3.1). It is concluded that Ca supplementation of the usual diet in young adults suppresses bone resorption over a 2-week period. If sustained, this could result in suppression of the bone remodelling rate and an increase in bone mass over time. The findings of this short-term study with a relatively small number of young adults highlight the need for a longer-term intervention study of the effect of increased Ca intake on bone mass in this age group.

The effect of dietary sodium intake on biochemical markers of bone metabolism in young women.

To investigate the effect of a low (80 mmol/d) or high (180 mmol/d) Na intake for 14d on biochemical markers of bone turnover in Na-sensitive and Na-non-sensitive healthy young women, twenty-nine subjects were screened for responsiveness of urinary Ca excretion to increasing dietary Na intake (40, 80, 120 and 200 mmol/d for 7 d). In a crossover study, the eight Na-sensitive and eight of the twenty-one Na-non-sensitive subjects were randomly assigned to diets containing either 80 or 180 mmol Na/d for 14d followed by crossover to the alternative diet for a further 14 d. Dietary Ca was restricted to 12.5 mmol/d throughout. During each dietary period, fasting morning first void urine samples (last 3 d) and fasting blood serum samples (morning of twelfth day) were collected. Increasing Na intake from 80 to 180 mmol/d increased urinary Na about twofold in both the Na-sensitive and Na-non-sensitive groups and increased urinary Ca excretion (by 73%) in the Na-sensitive group only. Biochemical markers of bone resorption (urinary pyridinoline and deoxypyridinoline) and bone formation (serum osteocalcin and bone-specific alkaline phosphatase; EC 3.1.3.1) were unaffected by increasing dietary Na in either group. It is concluded that the Na-induced calciuria observed in the Na-sensitive healthy young women did not result in increased bone resorption or turnover and, despite restricted Ca intake, adaptation of dietary Ca absorption may have compensated for the increased urinary Ca loss.

Measurement of intracellular calcium levels of human spermatozoa acrosome reacted by electropermeabilization.

Human spermatozoa readily undergo in vitro capacitation and the acrosome reaction when exposed to a single, brief, high-voltage electric pulse in a medium containing calcium. Using the fluorescent calcium indicator, Fura-2, the changes in the intracellular calcium concentration of human sperm following exposure to voltages in the range of 200-2000 V cm-1 were measured. The induction of the acrosome reaction by the electropermeabilization pulse was also monitored. In comparison, changes in calcium uptake during a standard capacitation procedure in which sperm samples were incubated for 7 h were analyzed. At hourly intervals, acrosome reaction induction and intracellular calcium uptake were measured. The results indicate that electropermeabilization rapidly and efficiently produces high populations of acrosome-reacted sperm and a corresponding and associated increase in the intracellular calcium concentration.