Nitrogen metabolism and bone metabolism markers in healthy adults during 16 weeks of bed rest.

BACKGROUND: The associations between nitrogen metabolism and bone turnover during bed rest are still not completely understood. METHODS: We measured nitrogen balance (nitrogen intake minus urinary nitrogen excretion) and biochemical metabolic markers of calcium and bone turnover in six males before head-down tilt bed rest (baseline), during 2, 10, and 14 weeks of immobilization, and after reambulation. RESULTS: The changes in nitrogen balance were highest between baseline and week 2 (net change, -5.05 +/- 1.30 g/day; 3.6 +/- 0.6 g/day at baseline vs -1.45 +/- 1.3 g/day at week 2; P<0.05). In parallel, serum intact osteocalcin (a marker of bone formation) was already reduced and renal calcium and phosphorus excretions were increased at week 2 (P <0.05). Fasting serum calcium and phosphorus values and renal excretion of N-telopeptide (a bone resorption marker) were enhanced at weeks 10 and 14 (P <0.05-0.001), whereas serum concentrations of parathyroid hormone, calcitriol, and type I collagen propeptide (a marker of bone collagen formation) were decreased at week 14 (P <0.05-0.01). Significant associations were present between changes of serum intact osteocalcin and 24-h calcium excretion (P <0.001), nitrogen balance and 24-h phosphorus excretion (P <0.001), nitrogen balance and renal N-telopeptide excretion (P <0.05), and between serum osteocalcin and nitrogen balance (P <0.025). CONCLUSIONS: Bone formation decreases rapidly during immobilization in parallel with a higher renal excretion of intestinally absorbed calcium. These changes appear in association with the onset of a negative nitrogen balance, but decreased bone collagen synthesis and enhanced collagen breakdown occur after a time lag of several weeks.

Microgravity inhibits intestinal calcium absorption as shown by a stable strontium test.

BACKGROUND: Little is known about the onset and degree of biochemical and functional alterations in calcium metabolism during microgravity. OBJECTIVE: To evaluate the effect of microgravity on intestinal calcium absorption and calcium-regulating hormones under metabolic ward conditions. MATERIALS AND METHODS: Fractional calcium absorption (Fc240 in percentage of dose administered) was determined pre-flight, in-flight and post-flight, by use of a stable strontium test in one cosmonaut who spent 20 days in space. Moreover, a sequence of blood samples was collected for the determination of serum parathyroid hormone (PTH), 25-hydroxyvitamin D, calcitriol and serum C-telopeptide (CTx, biomarker of bone resorption) levels. During all periods of data collection, calcium intake was held constant at a minimum level of 1.000 mg day(-1) and a daily supplement of 16.6 microg vitamin D2 was given. Personal ultraviolet (UV) light exposure was measured during the whole mission using a biologically weighting UV dosimeter. RESULTS: Fc240 was markedly reduced on flight day 19 (4.4%) as compared to pre-flight and post-flight data (13.4% and 17.2%, respectively). Serum calcitriol levels fell from 40.6 pg mL(-1) (mean pre-flight level) to 1.3 pg mL(-1) on flight day 18 and returned into the normal range after recovery. Serum CTx increased during the flight, while serum PTH and 25-hydroxyvitamin D levels did not change significantly. CONCLUSIONS: Intestinal calcium absorption can be diminished after only three weeks of microgravity. Changes are associated with a severe suppression of circulating calcitriol levels, but are independent of exogenous vitamin D supply and serum PTH levels.

Exercise-trained young men have higher calcium absorption rates and plasma calcitriol levels compared with age-matched sedentary controls.

The effect of physical activity on human calcium (Ca) metabolism is still not completely understood. Thus, we investigated fractional Ca absorption using a stable strontium test (Fc(240)), calciotropic hormones, and renal Ca excretion in 31 young men with a high activity level (GH) and in 26 age-matched sedentary control subjects (GL). Weekly hours spent on physical activity, obtained with a questionnaire were 15.0 +/- 6.6 (GH) and 1.0 +/- 1.4 (GL), respectively. Serum testosterone levels were significantly lower in GH compared with GL (P < 0.005). Dietary Ca intake (4-day food record) was twice as high in GH compared with GL men (P < 0.001). GH had significantly higher serum calcitriol levels and Fc(240) values than GL (P < 0.001 and P < 0.01, respectively). In a stepwise multiple regression analysis including serum levels of 25-hydroxyvitamin D, calcitriol, testosterone, and dietary Ca intake, only calcitriol was significantly correlated with Fc(240) (P = 0. 017). Twenty-four hour renal Ca excretion was only slightly higher in GH compared with GL (P < 0.05). However, additional Ca losses might have occurred through the extensive sweating of GH, as indicated by a difference of 1.7 liter between fluid intake and renal fluid excretion (P < 0.001). In summary, we observed a higher fractional Ca absorption rate in physically active young men compared with sedentary controls which is probably mediated by calcitriol. The low testosterone serum levels of the athletes were obviously not a limiting factor in Ca absorption efficiency. An additional Ca retention might, however, only be obtained if absorbed Ca exceeded total obligatory Ca losses.

Lactose does not enhance calcium bioavailability in lactose-tolerant, healthy adults.

BACKGROUND: There is evidence from animal studies that lactose has a beneficial effect on intestinal calcium absorption. However, data concerning the effect of lactose on calcium absorption in lactose-tolerant adults are inconclusive. OBJECTIVE: Our objective was to investigate the effect of lactose on calcium bioavailability in humans by the use of a stable-strontium test under controlled metabolic conditions. DESIGN: Eleven healthy, lactose-tolerant subjects (8 women, 3 men) randomly received a bolus of 2.27 mmol strontium alone (load A), the bolus with 35 g lactose (load B), or the bolus with 17.5 g glucose and 17.5 g galactose (load C). Blood samples were drawn at 0, 15, 30, 60, 90, 180, 240, and 300 min. Urine specimens were collected during the time intervals -2 to 0, 0-2, 2-4, 4-6, and 6-24 h. RESULTS: Pharmacokinetic parameters of strontium bioavailability were comparable for all 3 loads. In detail, fractional absorption at 240 min for loads A, B, and C was 12.1 +/- 0.7%, 13.0 +/- 1.1%, and 12.2 +/- 0.7%, respectively. Areas under the curve for 0-240 min were 70.8 +/- 6.3, 69.6 +/- 3.5, and 65.8 +/- 5.1 micromol*h/L for loads A, B, and C, respectively (NS). Moreover, fractional strontium excretion values of 5.1 +/- 0.8% (load A), 5.8 +/- 0.4% (load B), and 5.2 +/- 0.8% (load C) were not significantly different. CONCLUSIONS: Lactose does not have a beneficial effect on calcium bioavailability in lactose-tolerant adults.

Physiologic fluctuations of serum estradiol levels influence biochemical markers of bone resorption in young women.

We investigated the effect of physiologic variations in sex hormone levels during the menstrual cycle on biomarkers of bone turnover. Blood and 24-h and fasting urine samples were obtained in nine women (age, 25.1+/-3.0 yr) with regular menstrual cycles during the early follicular period (t1), 3 days before ovulation (t2), 3 days after ovulation (t3), at the midluteal period (t4) and again during the early follicular period of the next cycle (t5). All subjects had a calcium intake covering current dietary recommendations (above 1,000 mg/day, standardized food record). Serum calcium, phosphorus, calcitriol, 24-h and 2-h fasting urinary calcium, and phosphorus excretion remained constant during the menstrual cycle. Serum 25-hydroxyvitamin D3 levels decreased slightly from the beginning until the end of the study (P<0.05), indicating low cutaneous vitamin D synthesis during wintertime. The serum levels of sex hormones showed typical monthly variations, with the lowest estradiol (E2) levels at t1 and t5. Fasting 2-h pyridinoline (Pyd) concentrations (a marker of bone resorption) fell from t1 to t3 and rose again at t5 (P<0.01). Similar variations were observed for the resorption marker deoxypyridinoline (Dpd; P<0.05). The amplitude of the two biomarkers was 32% and 33%, respectively. The serum levels of the carboxyterminal propeptide of type I collagen (a marker of bone formation) showed an inverse cyclic pattern, as compared with the pyridinium cross-links. Low concentrations were observed at t1; a rise occurred until t3 and was followed by a decrease until t5 (P<0.05). A similar cyclic pattern was observed for serum PTH levels, with the highest concentrations at t3 (P<0.05). Dpd and Pyd values were significantly correlated with serum E2 levels (r = 0.52; P<0.0001 and r = 0.50; P<0.001, respectively). Neither progesterone nor LH nor FSH was correlated with Pyd or Dpd levels. The data suggest that normal menstrual cycling in young women is associated with monthly fluctuations in bone turnover. This physiological effect of the menstrual cycle is most probably related to variations in serum E2 concentrations.

Wheat bran supplementation does not affect biochemical markers of bone turnover in young adult women with recommended calcium intake.

We investigated the effect of wheat bran on biochemical indicators of Ca and bone metabolism in nineteen healthy women, aged 25.5 (SE 0.9) years. Subjects received six wheat bran biscuits or six white flour biscuits per day for a period of 4 weeks (crossover). Wheat bran consumption increased fibre intake from 17.7 (SE 1.3) to 29.6 (SE 1.3) g/d (7 d food record) and enhanced P intake from 1225 (SE 59) mg/d to 1663 (SE 65) mg/d; P < 0.001. Mean daily Ca intake during wheat bran consumption (1110 (SE 82) mg/d) significantly (P = 0.008) exceeded Ca ingestion during the white flour period (955 (SE 67) mg/d). Wheat bran increased the number of defecations per week from 7.9 (SE 0.8) to 12.2 (SE 1.4) (P = 0.0018). Urinary Ca excretion over 24 h significantly (P = 0.021) decreased from 473 (SE 53) mumol/mmol creatinine (control period) to 339 (SE 37) mumol/mmol creatinine (wheat bran period). Serum 25-hydroxyvitamin D, 2 h fasting urinary Ca/creatinine excretions and 24 h urinary P excretion remained constant. No differences in serum levels of carboxy-terminal propeptide of type I procollagen (biomarker of bone formation) or in 2 h fasting urinary hydroxyproline/creatinine excretions (biomarker of bone resorption) were observed at the end of the two cycles of dietary supplementation. We conclude that a high fibre intake of approximately 30 g/d has no significant adverse effects on bone turnover in subjects with Ca intakes above 1000 mg/d and that the reduction in 24 h urinary Ca excretion is most probably the result of an adaptation process, induced by a decrease in net absorbed Ca.

Seasonal variations in vitamin D status and calcium absorption do not influence bone turnover in young women.

OBJECTIVE: To evaluate the effect of seasonal variations in UV B-exposure on calcium absorption and bone turnover in young women with the overall goal to assess the potential benefit of a vitamin D supplementation during wintertime. DESIGN: Cross-sectional study. SETTING: Area of Bonn, Germany (51 degrees N). SUBJECTS: Thirty-eight women (24.5+/-0.5 y) studied in winter and 38 females of the same age (24.7+/-0.4 y) studied in summer. RESULTS: As estimated by a 4 d food record, both groups had similar dietary calcium and phosphorus intakes (> 1200 mg/d, respectively) covering actual recommendations. Significant reductions in serum concentrations of 25-hydroxyvitamin D (25OHD) and calcitriol, fractional calcium absorption (Fc220, measured by means of a stable strontium test), 24h urinary calcium and 24h urinary phosphorus excretion were observed during wintertime. 25OHD but not calcitriol was correlated with Fc220 values and with 24h urinary phosphorus excretion. Moreover, Fc220 was related to 24 h urinary calcium excretion. Fasting 2 h-urinary deoxypyridinoline concentrations (biomarker of bone resorption) and serum levels of carboxyterminal propeptide of type I procollagen (biomarker of bone formation) showed no differences between summer and winter. CONCLUSIONS: Our data indicate a decrease in intestinal calcium and phosphorus absorption during wintertime, most likely because of a reduction in serum 25OHD levels. Since bone turnover was not affected by the seasonal differences in mineral metabolism, there is no objective for young women with high calcium intake to supplement vitamin D during wintertime.

Crystallizable HIV-1 protease derived from expression of the viral pol gene in Escherichia coli.

A plasmid vector was used to express the HIV-1 pol open reading frame under the regulation of the bacterial trp promoter in Escherichia coli. This expression system has been used as a source of recombinant viral protease. The self-processed active enzyme was recovered from a soluble fraction of a bacterial cell lysate and purified by a procedure involving four steps of chromatography. The protocol yielded 0.3 mg of protease for each liter of bacterial culture. The protease formed tetragonal bipyramidal crystals which have been used in high-resolution X-ray diffraction studies.

Expression and localization of myelin basic protein in oligodendrocytes and transfected fibroblasts.

Myelin basic protein (MBP) is a major structural component of myelin. It is expressed exclusively in myelinating glia (oligodendrocytes in the CNS and Schwann cells in the PNS) and is localized to the cytoplasmic surface of the plasma membrane and myelin membrane produced by these cells. The work described here concerns the mechanism of plasma membrane localization of MBP in myelinating glial cells and whether it involves differentiated functions specific to these cells or general functions of plasma membrane assembly common to all cells. To this end, the subcellular localization of endogenous MBP in mouse oligodendrocytes was compared with that of transiently expressed MBP in monkey fibroblasts (Cos-1 cells) transfected with an MBP expression vector containing cDNA for rat 14K MBP. The steady-state levels of MBP-specific RNA and of MBP polypeptide expressed in the transfected fibroblasts were comparable to the levels expressed in oligodendrocytes in primary culture. MBP localization was analyzed in whole cells by immunofluorescence and in specific intracellular compartments by subcellular fractionation. The results show that MBP expressed in wild-type oligodendrocytes is localized to the plasma membrane. In contrast, MBP expressed in transfected fibroblasts appears dispersed in the cytoplasm and is distributed uniformly among the various subcellular fractions.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and expression of myelin basic protein gene sequences in the mld mutant mouse: reiteration and rearrangement of the MBP gene.

The mld mutation on chromosome 18 in the mouse is a putative allele of the shiverer (shi) mutation. We have analyzed the structure of myelin basic protein (MBP) gene sequences in mld DNA by restriction mapping of genomic DNA. The results indicate that the mld chromosome carries two copies of the MBP structural gene, one of which is intact and one of which is interrupted. Genetic analysis indicates that the interrupted gene is close to the intact MBP structural gene and cosegregates with the mld mutation. We have also analyzed the levels of MBP polypeptides and MBP-specific mRNA in wild-type, homozygous and heterozygous shiverer and mld mice and in mice carrying both mutations. The results indicate that both shi and mld are cis-acting codominant mutations that cause severely reduced steady state levels of MBP-specific mRNA and MBP polypeptides in the brain. We have analyzed the total number of oligodendrocytes and the number of MBP-positive oligodendrocytes in mld and shi brain primary cultures. In shi cultures, none of the oligodendrocytes expresses MBP. However, in mld cultures, approximately 5% of the oligodendrocytes express MBP. The nature of the "revertant" mld oligodendrocytes is not known.