Absorption, Pharmacokinetics, and Urinary Excretion of Pyridines After Consumption of Coffee and Cocoa-Based Products Containing Coffee in a Repeated Dose, Crossover Human Intervention Study.

SCOPE: The present study assesses the absorption, pharmacokinetics, and urinary excretion of coffee pyridines and their metabolites after daily regular exposure to specific dosages of coffee or cocoa-based products containing coffee (CBPCC), considering different patterns of consumption. METHODS AND RESULTS: In a three-arm, crossover, randomized trial, 21 volunteers are requested to randomly consume for 1 month: one cup of espresso coffee per day, three cups of espresso coffee per day, or one cup of espresso coffee plus two CBPCC twice per day. The last day of the one-month treatment, blood and urine samples are collected for 24 h. Trigonelline, N-methylpyridinium, N-methylnicotinamide, and N-methyl-4-pyridone-5-carboxamide are quantified. Trigonelline and N-methylpyridinium absorption curves and 24-h urinary excretion reflect the daily consumption of different servings of coffee or CBPCC, showing also significant differences in main pharmacokinetic parameters. Moreover, inter-subject variability due to sex and smoking is assessed, showing sex-related differences in the metabolism of trigonelline and smoking-related ones for N-methylpyridinium. CONCLUSION: The daily exposure to coffee pyridines after consumption of different coffee dosages in a real-life setting is established. This data will be useful for future studies aiming at evaluating the bioactivity of coffee-derived circulating metabolites in cell experiments, mimicking more realistic experimental conditions.

Consumption of a dark roast coffee decreases the level of spontaneous DNA strand breaks: a randomized controlled trial.

PURPOSE: Coffee consumption has been reported to decrease oxidative damage in peripheral white blood cells (WBC). However, effects on the level of spontaneous DNA strand breaks, a well established marker of health risk, have not been specifically reported yet. We analyzed the impact of consuming a dark roast coffee blend on the level of spontaneous DNA strand breaks. METHODS: Healthy men (n = 84) were randomized to consume daily for 4 weeks either 750 ml of fresh coffee brew or 750 ml of water, subsequent to a run in washout phase of 4 weeks. The study coffee was a blend providing high amounts of both caffeoylquinic acids (10.18 ± 0.33 mg/g) and the roast product N-methylpyridinium (1.10 ± 0.05 mg/g). Before and after the coffee/water consumption phase, spontaneous strand breaks were determined by comet assay. RESULTS: At baseline, both groups exhibited a similar level of spontaneous DNA strand breaks. In the intervention phase, spontaneous DNA strand breaks slightly increased in the control (water only) group whereas they significantly decreased in the coffee group, leading to a 27% difference within both arms (p = 0.0002). Food frequency questionnaires indicated no differences in the overall diet between groups, and mean body weight during the intervention phases remained stable. The consumption of the study coffee substantially lowered the level of spontaneous DNA strand breaks in WBC. CONCLUSION: We conclude that regular coffee consumption contributes to DNA integrity.

Urinary N-methylpyridinium and trigonelline as candidate dietary biomarkers of coffee consumption.

SCOPE: In order to validate the in vivo function of putatively healthy molecules in foods, human intervention studies are required. As the subject's compliance concerning intake or abstinence of a given food is considered mandatory to be monitored by biomarkers, the objective was to identify analytical markers for coffee consumption. METHODS AND RESULTS: Urine samples collected from coffee drinkers were compared with those of non-coffee drinkers using hydrophilic liquid interaction chromatography (HILIC)/time-of-flight mass spectrometry-based metabolite profiling. Two urinary molecules, found to be contributing most to the dissimilarities between both groups, were identified as N-methylpyridinium (NMP) and trigonelline and their suitability as coffee-specific biomarkers was validated by means of a coffee intervention study. After the volunteers (five females and four males) consumed a single dose of coffee, morning urine was collected for 10 days while staying abstinent from any coffee. HILIC-MS/MS-stable isotope dilution analysis (SIDA) revealed elevated urinary concentrations of trigonelline and NMP for up to 48 (p=0.001) and 72 h (p=0.002), respectively, after coffee consumption when compared with non-coffee drinkers. CONCLUSION: Analysis of urinary NMP allows to check for coffee consumption within a period of 3 days and is proposed as a dietary biomarker which might be used as an analytical probe to control compliance in human intervention studies on coffee.

Identification of two novel metabolites of SCH 486757, a nociceptin/orphanin FQ peptide receptor agonist, in humans.

The study of human metabolism of endo-8[bis(2-chlorophenyl)methyl]-3-(2-pyrimidinyl)-8-azabicyclo[3.2.1]octan-3-ol (SCH 486757) after a 200-mg oral dose of the drug to healthy volunteers in the first-in-human study is presented. The structural elucidation of two unique metabolites, which were detected in the process of metabolite characterization in human plasma and urine by liquid chromatography-mass spectrometry (LC-MS), is described. These metabolites (M27 and M34) were initially detected in human plasma at high levels (>35% of the LC-MS response of the parent drug). Additional LC-MS experiments (hydrogen/deuterium exchange and accurate mass measurement) were used to determine structures of metabolites. It was found that both metabolites were formed through a loss of the C-C bridge from the tropane moiety with the conversion into a substituted pyridinium compound. This metabolic process has not been reported previously. Because of the apparent high abundance of metabolites based on the LC-MS response, actual circulating amounts of these metabolites relative to the parent drug were determined semiquantitatively to evaluate their coverage in preclinical species. With the use of reference standards, it was shown that the LC-MS response of M27 and M34 in human plasma was much higher than that of the parent compound. Actual amounts of M27 and M34 metabolites were less than 5% of the level of the parent drug; therefore, additional assessment was not required.

Identification of urinary modified nucleosides and ribosylated metabolites in humans via combined ESI-FTICR MS and ESI-IT MS analysis.

The physiological response of the human body to several diseases can be reflected by the metabolite pattern in biological fluids. Cancer, like other diseases accompanied by metabolic disorders, causes characteristic effects on cell turnover rate, activity of modifying enzymes, and RNA/DNA modifications. This results in an altered excretion of modified nucleosides and biochemically related compounds. In the course of our metabolic profiling project, we screened 24-h urine of patients suffering from lung, rectal, or head and neck cancer for previously unknown ribosylated metabolites. Therefore, we developed a sample preparation procedure based on boronate affinity chromatography followed by additional prepurification with preparative TLC. The isolated metabolites were analyzed by ion trap mass spectrometry (IT MS) and Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). IT MS was applied for LC-auto MS(3) screening runs and MS(n(n=4-6)) syringe pump infusion experiments, yielding characteristic fragmentation patterns. FTICR MS measurements enabled the calculation of corresponding molecular formulae based on accurate mass determination (mass accuracy: 1-5 ppm for external and sub-ppm values for internal calibration). We were able to identify 22 metabolites deriving from cellular RNA metabolism and related metabolic pathways like histidine metabolism, purine biosynthesis, methionine/polyamine cycle, and nicotinate/nicotinamide metabolism. The compounds 1-ribosyl-3-hydroxypyridinium, 1-ribosyl-pyridinium, and 3-ribosyl-1-methyl-l-histidinium as well as a series of ribosylated histamines, conjugated to carboxylic acids at the N(omega)-position were found as novel urinary constituents. The occurrence of the modified nucleosides 2-methylthio-N(6)-(cis-hydroxyisopentenyl)-adenosine, 5-methoxycarbonylmethyl-2-thiouridine, N(6)-methyl-N(6)-threonylcarbamoyladenosine, and 2-methylthio-N(6)-threonylcarbamoyladenosine in human urine is verified for the first time.

[Metabolic bone disease in premature infants and genetic polymorphisms]. / Koraszülöttek csontanyagcsere-betegsége és genetikai polimorfizmusok.

UNLABELLED: Metabolic bone disease is an important complication among infants very-low-birth-weight (< 1500 g). In adults, osteoporosis has been shown to be associated with polymorphisms of vitamin D receptor, estrogen receptor, and collagen Ialpha1 receptor genes. AIM: The primary goal of the study was to investigate the possible association between metabolic bone disease and the allelic polymorphisms of these three genes. METHOD: 104 infants very-low-birth-weight were enrolled to the study. Bone formation (serum alkaline phosphatase, osteocalcin) and bone resorption (urinary excretion of calcium and pyridinium crosslink) markers were determined and x-rays of the chest and wrist (together with the distal portions of associated long bones) were obtained. RESULTS: Thirty infants (28,8%) were diagnosed with metabolic bone disease based on high activity of bone formation, bone resorption markers, and positive radiologic signs. Statistically significant correlation between thymine-adenine repeat [(TA) n ] allelic variant of estrogen receptor gene and bone disease was observed. Infants with metabolic bone disease more often carried low number of repeats [(TA) n < 19] [odds ratio (OR): 5.82, 95% confidence interval (CI): 2.26-14.98]. Significantly higher number of repeats [(TA)n > 18] was found more frequently in the control group (OR: 0.20, 95% CI: 0.05-0.82). Furthermore significant interaction between vitamin D receptor and collagen Ialpha1 receptor genotypes ( p = 0.023) was observed. In a forward stepwise logistic regression model, bone disorder of preterms correlated with male gender ( p = 0.001), duration of hospitalization ( p = 0.007), homozygous allelic variants of high number of (TA) n repeats ( p = 0.025) and interaction between vitamin D receptor (Tt) and estrogen receptor (homozygous allelic variants of low number of repeats) genotype ( p = 0.037). CONCLUSION: The results suggest that the development of metabolic bone disease in infants very-low-birth-weight may be associated with genetic polymorphisms.

Prognostic value of urinary pyridinium crosslinks and their derivatives in multiple myeloma.

Bone disease is a common feature of multiple myeloma (MM). The goal of this study was to assess the prognostic significance of urinary markers of bone metabolism in MM. Urinary levels of total pyridinoline (T-Pyd), deoxypyridinoline (T-Dpd), crosslinked N-telopeptide (Ntx), C-telopeptide (Ctx) of type I collagen and immunologic free deoxypyridinoline (f-Dpd) were assessed in 82 consecutive, previously untreated MM patients (aged 65-75 years) diagnosed between June 1995 and December 1998. A correlation between disease stage according to the Durie-Salmon classification and T-Pyd (p=0.034) and T-Dpd (p=0.007) was observed, while T-Pyd (p=0.015) and to a lesser extent f-Dpd (p=0.081) were correlated to bone involvement measured by plain X-ray. None were correlated to M-component or magnetic resonance imaging (MRI). In univariate analysis high T-Pyd (p=0.007), T-Dpd (p=0.027), f-Dpd (p<0.001), and Ctx (p=0.011) were associated with shorter overall survival, whereas only f-Dpd (p=0.0025) was associated with a shorter disease-free survival. In multivariate analysis, C-reactive protein and f-Dpd were independent prognostic factors for overall survival. This retrospective analysis defined new independent prognostic indicators of survival in patients with newly diagnosed myeloma. Indeed, urinary markers of bone resorption can easily be measured at diagnosis and have independent prognostic significance to refine the prognosis of MM patients.

Comparison of spot tests with AdultaCheck 6 and Intect 7 urine test strips for detecting the presence of adulterants in urine specimens.

BACKGROUND: Several adulterants are used to mask tests for abused drugs in urine. Adulterants such as "Klear" and "Whizzies" contain potassium nitrite while "Urine Luck" contains pyridinium chlorochromate (PCC). The presence of these adulterants cannot be detected by routine specimen integrity check (pH, specific gravity, creatinine and temperature). We previously reported the development of rapid spot tests to detect the presence of these adulterants. AdultaCheck 6 and Intect 7 urine test strips are commercially available for detecting the presence of these adulterants along with specific gravity, creatinine and pH in urine. METHODS: The performance of these two test strips for detecting adulterants was compared with the results obtained by spot tests. RESULTS: Both AdultaCheck 6 and Intect 7 effectively detected the presence of nitrite and pyridinium chlorochromate in urine. Moreover, both test strips successfully detected the presence of glutaraldehyde, for which no spot test is currently available. High amount of glucose and ascorbic acid did not cause any false positive result with AdultaCheck 6 or Intect 7. CONCLUSIONS: Both AdultaCheck 6 and Intect 7 can be used for checking the integrity of a urine specimen submitted for drugs of abuse testing.

Variability of collagen crosslinks: impact of sample collection period.

Because of the variability of collagen crosslinks, their use as markers for bone resorption is often criticized. We hypothesized that the variability could be reduced by collecting urine for 24 hours (or longer) instead of using single voids, and by not normalizing to creatinine. Urine samples were collected from 22 healthy subjects during two or more 24-hour periods. Each 24-hour pool and each 2nd void of the day were analyzed for N-telopeptide (NTX), pyridinium (PYD), and deoxypyridinoline (DPD) crosslinks. Data were analyzed by using linear regression. For NTX, R2 for the two, 2nd-void samples (n = 38) was 0.55, whereas R2 for the two 24-hour pools was 0.51 or 0.52, expressed per day or per creatinine. For PYD and DPD, R2 for the 2nd-void samples was 0.26 and 0.18, R2 for the 24-hour pools expressed per day was 0.58 and 0.74, and R2 for the 24-hour pools expressed per creatinine was 0.65 and 0.76, respectively. Regression of the 2nd void and the corresponding 24-hour pool, expressed per day, yielded R2 = 0.19, 0.19, and 0.08, for NTX, PYD, and DPD, respectively (n = 76 each). For the 2nd-void sample and its corresponding 24-hour pool, expressed per creatinine, R2 = 0.24, 0.33, and 0.08, respectively. In a separate study, the coefficient of variation for NTX was reduced (P < 0.05) when data from more than one 24-hour collection were combined. Thus, the variability inherent in crosslink determinations can be reduced by collecting urine for longer periods. In research studies, the high variability of single-void collections, compounded by creatinine normalization, may alter or obscure findings.

The ovariectomized sheep as a model for human bone loss.

The aim of this study was to determine the effects of ovariectomy on the trabeculae of ovine iliac bone, with a view to the further development of a model for human osteoporosis. Seven ovariectomized (OVX) and five control Dorset ewes were observed for one year. Iliac crest bone samples were obtained at the time of ovariectomy ("time 0") and again when the animals were killed 12 months later. At "time 0" and at 3-month intervals thereafter samples of blood and urine were collected for the assay of serum 17beta-oestradiol and osteocalcin (OC) and urinary deoxypyridinium (D-PYR). One year after ovariectomy, trabecular bone volume and thickness were reduced (P<0.05) and trabecular separation was increased (P<0.05) as compared with the controls. In OVX sheep, serum 17beta-oestradiol concentrations were significantly lower at the end of the experiment than at "time 0", while serum OC and urinary D-PYR concentrations were significantly higher (P<0.001). The results suggest that the OVX sheep is a valid model for changes in trabecular bone architecture associated with oestrogen deficiency, especially in women experiencing early menopause.

Urine products of bone breakdown as markers of bone resorption and clinical usefulness of urinary hydroxyproline: an overview.

PURPOSE: The purpose of this study is to review the urine products of bone breakdown as markers of bone resorption and usefulness of urinary hydroxyproline. DATA: Related researches published in 1985 - 2000 were systematically reviewed. RESULTS: Bone markers could be used for early diagnosis of bone metabolic diseases. Biochemical markers of bone resorption that reflect osteoclast activity and/or collagen degradation provide a new and potentially important clinical tool for the assessment and monitoring of bone metabolism. Assessment of bone resorption can be achieved with measurement of urinary hydroxylysine glycosides, urinary excretion of the collagen pyridinium cross-links, urinary excretion of type I collagen telopeptide breakdown products (cross-linked telopeptides) and urinary hydroxyproline. CONCLUSION: Urinary hydroxyproline has been in use as a marker of bone resorption, but it lacks sensitivity and specificity. It is a modified amino acid that is a metabolic product of collagen breakdown. Hydroxyproline may be released either free or with fragments of the collagen molecule attached during bone resorption, and it is also liberated by the breakdown of complement and nonskeletal collagen.

[Collagen breakdown in ankylosing spondylitis-relationship to disease related parameters]. / Kollagenabbauaktivität bei Spondylitis ankylosans in Beziehung zu krankheitsrelevanten Daten.

OBJECTIVE: Patients with ankylosing spondylitis (AS) often develop osteoporosis particularly of the axis skeleton. To investigate disease-related or therapeutic influence on bone catabolism, we quantified the total excretion of the collagen crosslinks (CL) pyridinoline (Pyd) and deoxypyridinoline (Dpyd) in 91 AS patients (26 f, 65 m) in relation to disease activity or stage and therapy with NSAID. METHODS: CL were determined by HPLC (High Performance Liquid Chromatography). RESULTS: The AS patients show a highly significant positive correlation between Pyd and the inflammatory activity (CrP, r = 0.36, p < 0.001: ESR, r = 0.379, p < 0.001). Also the quotient Pyd/Dpyd correlates positively to the inflammatory activity (CrP, r = 0.262, p < 0.001: ESR, r = 0.325, p < 0.002). In the case of increased inflammatory disease activity (CrP > or = 10 mg/l vs CrP < 10 mg/l or ESR > or = 10 30 mm vs ESR < 30 mm), Pyd excretion is raised significantly (p < 0.042 for CrP and p < 0.009 for ESR). Among those patients treated with NSAID therapy, significantly reduced levels for Dpyd (p < 0.001) and raised levels for the quotient Pyd/Dpyd (p < 0.002) appear. In the case of advanced radiological changes with evidence of syndesmophytes, Pyd (p = 0.014) and Dpyd (p < 0.004) were significantly raised in urine. Regarding the movement function (finger-floor distance, schober test), no significant correlation to crosslink excretion could be proven. CONCLUSION: From our investigations, we assume that osteoporosis in AS is primarily caused by an inflammatory-mediated degradation of bone.

Bone resorption in anorexia nervosa and rehabilitated patients.

OBJECTIVE: To assess the impact of anorexia nervosa and that of nutritional rehabilitation on bone resorption. DESIGN: Cross-sectional, observational study. SETTING: Rome, Italy SUBJECTS: Twenty-eight female patients affected by anorexia nervosa (AN, BMIor=18.5 kg/m(2)) and 34 age- and sex-matched healthy controls (CO, BMI >or=18.5 kg/m(2)). Among AN patients, 16 were affected by the 'restrictive' (ANr) and 12 by the 'purging' type (ANp) of anorexia nervosa. METHOD: Body weight, height and skeletal diameters were measured on each individual. The skeletal mass (SKM) was predicted from the skeletal diameters of the elbow, wrist, knee and ankle, using the equation of Martin. Twenty-four-hour urinary excretion of pyridinium crosslinks of collagen (pyridinoline (Pyd) and deoxypyridinoline (Dpd)) and creatinine was assessed by reversed-phase HPLC with fluorimetric detection after solid-phase extraction and by the Jaffé-method with deproteinization, respectively. RESULTS: Twenty-four-hour urinary output of Pyd and Dpd was not significantly different between AN and CO when expressed in absolute values, but AN showed higher bone resorption than CO when Pyd and Dpd excretion was adjusted by either creatinine (P<0.0000) or the SKM (P<0.05). Within the AN group, urinary excretion of both cross-links was significantly and consistently higher in ANp compared with ANr (P<0.05). However, these differences disappeared when crosslink output was adjusted either by urinary creatinine or SKM. RE subjects showed no differences in bone resorption with the AN group despite weight gain, being crosslink excretion consistently elevated compared to controls (Pyd: P<0.01 by creatinine and P<0.05 by SKM; Dpd: P<0.01 by creatinine and P<0.05 by SKM). CONCLUSION: Bone resorption is elevated in anorexia nervosa and different strategies for low-weight maintenance do not seem to have a differential impact. Increased bone resorption persists in subjects with past diagnosis of anorexia nervosa despite rehabilitation lasting more than 6 months. This finding indicates that bone mass and turnover should be monitored in anorexia nervosa patients and ex-patients well beyond recovery of normal body mass. Further investigation is warranted to examine the long-term effect of such prolonged increase in bone turnover at a young age.

Association between craniofacial growth and urinary bone metabolic markers (pyridinoline, deoxypyridinoline) in growing rats.

Pyridinoline (Pyr) and deoxypyridinoline (Dpyr) are intermolecular cross-links of mature collagen and reflect the bone turnover. The purpose of this study was to elucidate the association between craniofacial growth and urinary Pyr and Dpyr levels. Lateral cephalograms and 24-hour urine were taken for 7 male rats from 5 to 20 wks old. The urinary Pyr and Dpyr were quantified by high-performance liquid chromatography. The neurocranium and upper viscerocranium exhibited significant increases in size, with the maximum rate at around 6 wks old. The mandible presented more substantial growth, with the maximum change at 8 wks old. The urinary Pyr and Dpyr levels gradually increased and reached the maximum at 8 wks old. No prominent association was found between neurocranial growth and urinary levels of pyridinium cross-links, whereas Pyr and Dpyr levels exhibited similar time-dependent metabolic changes to mandibular growth. In conclusion, it is shown that urinary pyridinium cross-links may be useful for the prediction of mandibular growth.

The lumbar bone mineral density is affected by long-term poor metabolic control in adolescents with type 1 diabetes mellitus.

AIM: To analyze whether bone mineral density (BMD) and bone resorption status are influenced by long-term metabolic control and duration of disease in adolescents with long-standing type 1 diabetes mellitus. METHODS: Twenty-seven adolescents (age 13.1 +/- 1.7 years, duration of diabetes 6.9 +/- 3.0 years) were studied. The BMD, expressed as z score, was measured at the lumbar spine (L1-L4) using dual-energy X-ray absorptiometry, while the urinary excretion of total deoxypiridinoline (Dpyd), a marker of bone resorption, was measured by immunoassay and was corrected by creatinine (Cr). Linear and multivariate correlations between lumbar BMD z score or Dpyd/Cr excretion and age and disease variables [short-term (Hb A(1c latest)) or long-term (Hb A(1c whole duration)) metabolic control, duration, 'diabetes impact index' (mean Hb A(1c whole duration) x duration of disease in months)] were sought. RESULTS: In diabetic subjects the mean BMD z score was -0.44 +/- (SD) 1.02 (95% CI: -0.03; -0.84), and the Dpyd/Cr excretion was not increased. A negative correlation was found between lumbar BMD z score and age (r -0.59; p = 0.001), duration (r -0.39; p = 0.04), and the diabetes impact index (r -0.4; p = 0.04). The Dpyd/Cr ratio correlated negatively with age (r -0.40; p = 0.04) and positively with height velocity (r 0.42; p = 0.04). By using multiple linear regression, age showed a significant inverse correlation with lumbar BMD z score (beta = -0.39; p = 0.0005). A negative correlation was found between lumbar BMD z score and Hb A(1c whole duration) (beta = -0.40; p = 0.02) or diabetes impact index (beta = -0.001; p = 0.01). CONCLUSIONS: Poor metabolic control may expose adolescents with long-standing type 1 diabetes to the risk of developing osteopenia in adult age. Optimization of metabolic control in growing diabetic children may prevent osteoporosis in later life.

Urinary collagen cross-links as biochemical markers of growth: an evaluation of biological variables.

AIM: To investigate the validity of urinary pyridinium cross-links (pyridinoline and deoxypyridinoline) as markers of growth in healthy children. METHODS: Three pilot studies (P1-P3) were conducted to investigate the time of day, the minimal duration within a day, and how many times per week urine samples needed to be collected to obtain representative values of cross-link excretion in normal children 3-5 years of age. The results were used to design a 4-month longitudinal protocol to evaluate whether pyridinium cross-links could be used as markers of growth velocity. RESULTS: Mean differences from 24-hour values were only between 1 and 4% for urinary cross-links (nmol/h) in overnight 12-hour collections. Three consecutive collections were required for weekly output estimates with a maximum error of 10% in >90% of the children. During the 4-month longitudinal study, the regression equation of height velocity on pyridinoline and deoxypyridinoline excretion explained approximately 60% of the variance in the subgroup of subjects who provided three complete urinary collections per observation period. No relationship was observed when the cases with fewer or incomplete collections were included in the analysis. Cross-link values collected at baseline were of no use to predict height velocity at 4 months. CONCLUSIONS: Urinary pyridinium cross-links correlate with the growth velocity in healthy children when using an appropriate urinary collection protocol. However, their predictive value in this population is negligible.

The effect of 17beta-estradiol at doses of 0.5, 1 and 2 mg compared with placebo on early postmenopausal bone loss in hysterectomized women.

In a randomized double-masked placebo-controlled parallel-group trial 166 hysterectomized (+/- oophorectomy) perimenopausal and postmenopausal women aged 45-55 years with a follicle stimulating hormone level above 20 IU/l were treated with one daily dose of either 0.5 mg 17beta-estradiol (E2), 1 mg E2, 2 mg E2 or placebo for 2 years. Bone mineral density (BMD) and biochemical bone markers were determined. All three doses of E2 were significantly better than placebo with respect to change in BMD at the lumbar spine (L1-L4) (p<0.0001 for all pairwise comparisons) and hip (femoral neck, trochanter, Ward's triangle). The mean percentage change from baseline at the lumbar spine was -0.2%, 0.8% and 1.8% in the 0.5, 1 and 2 mg E2 groups respectively compared with -3.5% in the placebo group. Both 1 and 2 mg E2 were significantly better than placebo in increasing the BMD at the femoral neck (p<0.001), trochanter (p<0.01) and Ward's triangle (p<0.0001), while 0.5 mg E2 was significantly better than placebo at the femoral neck (p<0.001) and Ward's triangle (p<0.0001). The overall difference in mean percentage change in BMD at the femoral neck versus placebo (-0.2%) was 3.8% for 0.5 mg, 4.0% for 1 mg and 3.9% for 2 mg E2; the corresponding numbers for trochanter were 1.3%, 3.3% and 3.2%, respectively, and -2.2%,, 2.9, 2.9% and 4.0%, respectively, for Ward's triangle. More than half the women who received placebo presented with a decrease in BMD at the hip. The percentage of women in the 0.5 mg E2 group who maintained or increased BMD at the femoral neck, trochanter and Ward's triangle was 69%, 56% and 44%, respectively. For 1 mg E2 the numbers were 69%, 78% and 61% respectively, and for 2 mg E2 were 59%, 68% and 59% respectively. Osteocalcin, serum pyridinium crosslinks, urinary pyridinium cross-links and urinary hydroxyproline/creatinine decreased significantly (p<0.0001, p<0.05) in the 0.5, 1 and 2 mg E2 groups compared with the placebo group after 6 and 24 months of treatment.

Comparison of the humoral markers of bone turnover and bone mineral density in patients on haemodialysis and continuous ambulatory peritoneal dialysis.

Renal osteodystrophy is an important complication in patients with end-stage renal disease on maintenance dialysis. The aim of this study was to compare the biochemical markers of bone formation (serum collagen type I C-terminal propeptide) and resorption (serum deoxypyridinoline - DPD - and pyridinoline - PYR) with the bone mineral density (BMD) at lumbar spine, femoral neck, and forearm in patients with end-stage renal disease on haemodialysis (HD) versus continuous ambulatory peritoneal dialysis (CAPD). Fifty-nine adult patients, 45 on CAPD (18 females, 27 males) and 14 on HD (2 females, 12 males), were studied. The mean age was 44 +/- SEM 1.6 and 54.4 +/- 4.8 years, respectively. No significant differences in serum calcium, phosphorus, creatinine, and parathyroid hormone were found between patients on HD and CAPD in predialysis samples. Serum urea was significantly lower (p = 0.02) in the CAPD group. Serum PYR (nmol/l) and DPD (nmol/l) were significantly higher in patients on HD as compared with those on CAPD: 105 +/- 23.3 versus 43.7 +/- 3.47 (p = 0.007) and 31.0 +/- 2.4 versus 24.4 +/- 1.4 (p = 0.027), respectively. The results were still significantly higher in the HD patients following correction for serum creatinine and body mass index. There was a close correlation between dialysate DPD and creatinine in both dialysis modalities (HD r = 0.9, CAPD r = 0.76). The clearance of DPD did not differ significantly between the CAPD membrane and the HD membrane (p = 0.22). Serum collagen type I C-terminal propeptide was not significantly different between the HD and CAPD patients. The results were unaffected following correction for age and gender. The BMD was measured in 38 (65%) of the patients (HD n = 8, CAPD n = 30) by dual-energy X-ray absorptiometry and expressed as 'Z' scores. This was reduced at all sites in the patients with end-stage renal disease. The BMD was significantly lower at the ultradistal forearm (mostly trabecular bone) in HD patients as compared with CAPD patients (n = 0.02). A similar trend was observed at the lumbar spine, although the results failed to reach significance. In the whole population (n = 38), linear regression analysis revealed a significant negative correlation between BMD at the ultradistal forearm and serum PYR (r = -0.35, p = 0.04) and DPD (r = -0.33, p = 0.049). Combined measurements of BMD and biochemical markers of bone resorption may have potential in the identification of patients at high risk of bone loss who may require further evaluation of bone remodeling by bone histomorphometry.

Effect of estrogen deficiency on skeletal and alveolar bone density in sheep.

BACKGROUND: This study provides a longitudinal assessment of changes in alveolar and skeletal bone mineral density (BMD) in ovariectomized animals. METHODS: Following ovariectomy (OVX) (n = 6) or sham-operation (n = 6) intraoral radiographs were made at 4-month intervals and serum 17-beta-estradiol, osteocalcin, and interleukin (IL)-6, urinary deoxypyridinium, and salivary IL-6, deoxypyridinium, and osteocalcin concentrations were evaluated. Twelve months after surgery, animals were sacrificed and the mandible and radius/ulna removed. Bones were sectioned and radiographed. Mean BMD and cortical thicknesses were calculated from each region. RESULTS: OVX animals had a progressive decrease in serum 17-beta-estradiol, increased serum osteocalcin and IL-6, urinary deoxypyridinium and salivary IL-6, osteocalcin and deoxypyridinium (P < 0.001), suggesting that they were becoming osteoporotic. The BMD of the radius/ulna and mandibular alveolar bone was significantly reduced in OVX animals (P < 0.05 and P < 0.001, respectively). Reduced alveolar bone BMD became evident in OVX animals 6 months after surgery and became more severe during the subsequent 6 months. Alveolar crestal height was also significantly reduced in OVX animals (P < 0.001). These biochemical and density changes preceded a significant reduction in serum 17-beta-estradiol, which occurred between 4 and 8 months following surgery. CONCLUSIONS: Serial measurements of alveolar BMD predicts loss of skeletal BMD in OVX sheep. Changes in alveolar BMD precede estrogen deficiency, suggesting that early signs of reduced BMD may be detected in peri-menopausal women. The presence of biomarkers of bone metabolism within saliva and their correlation with reduced BMD suggests that saliva could be used as an adjunct screening method for assessment of skeletal bone density.

Urinary excretion of pyridinium crosslinks in short children treated with growth hormone.

The aim of this study was to evaluate the effect of growth hormone (GH) treatment on bone resorption in children with GH deficiency and those with idiopathic short stature. The study population included seven children with subnormal spontaneous GH secretion and 13 children with idiopathic short stature, all of them pre-pubertal. Anthropometric measurements, free, protein-bound and total urinary pyridinoline (Pyd) and deoxypyridinoline (Dpd), serum GH, and serum immunoreactive PTH were measured at baseline and months 1, 3, 6 and 12 of GH treatment. The urinary excretion of total Pyd and Dpd, standardized by the cube of height (m3) in overnight, 24-hour urine collections was not different from age-matched healthy controls at baseline in either group of patients. During treatment with human recombinant GH, both pyridinium crosslinks increased above normal values, reaching a peak after one month in children with GH deficiency and later (after 3-6 months) in children with short stature. Free and total crosslink forms were correlated, and GH treatment did not affect the proportion of free to bound crosslinks. Serum concentrations of iPTH showed a moderate but not statistically significant increase. This study provides no evidence of reduced bone resorption in untreated GH deficiency or in idiopathic short stature. GH treatment induced a marked, but temporary, increase of bone resorption in both groups of patients.