Absolute radiotracer concentration measurement using whole-body solid-state SPECT/CT technology: in vivo/in vitro validation.

The accuracy of recently approved quantitative clinical software was determined by comparing in vivo/in vitro measurements for a solid-state cadmium-zinc-telluride SPECT/CT (single photon emission computed tomography/x-ray computed tomography) camera. Bone SPECT/CT, including the pelvic region in the field of view, was performed on 16 patients using technetium-99m methylene diphosphonic acid as a radiotracer. After imaging, urine samples from each patient provided for the measurement of in vitro radiopharmaceutical concentrations. From the SPECT/CT images, three users measured in vivo radiotracer concentration and standardized uptake value (SUV) for the bladder using quantitative software (Q.Metrix, GE Healthcare). Linear regression was used to validate any in vivo/in vitro identity relations (ideally slope = 1, intercept = 0), within a 95% confidence interval (CI). Thirteen in vivo/in vitro pairs were available for further analysis, after rejecting two as clinically irrelevant (SUVs > 100 g/mL) and one as an outlier (via Cook's distance calculations). All linear regressions (R2 ≥ 0.85, P < 0.0001) provided identity in vivo/in vitro relations (95% CI), with SUV averages from all users giving a slope of 0.99 ± 0.25 and intercept of 0.14 ± 5.15 g/mL. The average in vivo/in vitro residual difference was < 20%. Solid-state SPECT/CT imaging can reliably provide in vivo urinary bladder radiotracer concentrations within approximately 20% accuracy. This practical, non-invasive, in vivo quantitation method can potentially improve diagnosis and assessment of response to treatment. Graphical abstract.

Liquid chromatography-mass spectrometry analysis of five bisphosphonates in equine urine and plasma.

Bisphosphonates are used in the management of skeletal disorder in humans and horses, with tiludronic acid being the first licensed veterinary medicine in the treatment of lameness associated with degenerative joint disease. Bisphosphonates are prohibited in horseracing according to Article 6 of the International Agreement on Breeding, Racing and Wagering (published by the International Federation of Horseracing Authorities). In order to control the use of bisphosphonates in equine sports, an effective method to detect the use of bisphosphonates is required. Bisphosphonates are difficult-to-detect drugs due to their hydrophilic properties. The complexity of equine matrices also added to their extraction difficulties. This study describes a method for the simultaneous detection of five bisphosphonates, namely alendronic acid, clodronic acid, ibandronic acid, risedronic acid and tiludronic acid, in equine urine and plasma. Bisphosphonates were first isolated from the sample matrices by solid-phase extractions, followed by methylation with trimethylsilyldiazomethane prior to liquid chromatography - tandem mass spectrometry analysis using selective reaction monitoring in the positive electrospray ionization mode. The five bisphosphonates could be detected at low ppb levels in 0.5mL equine plasma or urine with acceptable precision, fast instrumental turnaround time, and negligible matrix interferences. The method has also been applied to the excretion study of tiludronic acid in plasma and urine collected from a horse having been administered a single dose of tiludronic acid. The applicability and effectiveness of the method was demonstrated by the successful detection and confirmation of the presence of tiludronic acid in an overseas equine urine sample. To our knowledge, this is the first reported method in the successful screening and confirmation of five amino- and non-amino bisphosphonates in equine biological samples.

Quantitative analysis of bisphosphonates in biological samples.

Bisphosphonate drugs pose significant challenges for bioanalysis due to various complicating factors. In 2006, a novel approach, utilizing 'on-column' derivatization with diazomethane, was reported that revolutionized the application of liquid-chromatography-tandem mass spectrometry to bisphosphonates bioanalysis. The methodology enables superior biological sample clean-up while transforming bisphosphonates into species amenable to liquid-chromatography-tandem mass spectrometry detection. Since then, the approach has been successfully applied to numerous bisphosphonates. The use of an alternative methylation reagent - trimethylsilyl diazomethane - for on-column derivatization has been reported recently. This review focuses on published methods utilizing on-column derivatization for bioanalysis of major bisphosphonate drugs in biological matrices. Critical points required for successful application of on-column derivatization to the bioanalysis of bisphosphonates will be discussed.

Prolonged bisphosphonate release after treatment in women with osteoporosis. Relationship with bone turnover.

Bisphosphonates (BP), especially alendronate and risedronate, are the drugs most commonly used for osteoporosis treatment, being incorporated into the skeleton where they inhibit bone resorption and are thereafter slowly released during bone turnover. However, there are few data on the release of BP in patients who have received treatment with these drugs for osteoporosis. This information is essential for evaluating the possibility of BP cyclic therapy in these patients and for controlling their long-term presence in bone tissue. This study evaluated the urinary excretion of alendronate and risedronate in patients treated with these drugs for osteoporosis and analysed its relationship with bone turnover, time of previous drug exposure and time of treatment discontinuation. We included 43 women (aged 65±9.4 years) previously treated with alendronate (36) or risedronate (7) during a mean of 51±3 and 53±3 months, respectively, who had not been treated with other antiosteoporotic treatment and with a median time of discontinuation of 13.5 and 14 months, respectively. Both BP were detected in 24-hour urine by HPLC. In addition, bone formation (PINP) and resorption (NTx) markers were analysed. Both BP were also determined in a control group of women during treatment. Alendronate was detected in 41% of women previously treated with this drug whereas no patient previously treated with risedronate showed detectable urinary values. All control patients showed detectable values of both BP. In patients with detectable alendronate levels, the time of drug cessation was shorter than in patients with undetectable values (12 [6-19] versus 31 [7-72] months, p<0.001). Alendronate was not detected in any patient 19 months after treatment cessation. Alendronate levels were inversely related to time of treatment discontinuation (r=-0.403, p=0.01) and the latter was directly related to NTx (r=0.394, p=0.02). No relationship was observed with age, length of drug exposure, renal function or weight. In conclusion, contrary to risedronate, which was not detected in patients after cessation of treatment, alendronate was frequently detected in women previously treated with this agent up to 19 months after discontinuation of therapy. The relationship between alendronate levels and both bone resorption and time of treatment cessation further indicates a residual effect of this drug in bone, despite treatment discontinuation.

Capillary electrophoresis-electrochemiluminescence detection method for the analysis of ibandronate in drug formulations and human urine.

A simple, rapid and sensitive CE method coupled with electrochemiluminescence (ECL) detection for direct analysis of ibandronate (IBAN) has been developed. Using a buffer solution of 20 mM sodium phosphate (pH 9.0) and a voltage of 13.5 kV, separation of IBAN in a 30-cm length capillary was achieved in 3 min. ECL detection was performed with an indium tin oxide working electrode bias at 1.6 V (versus a Pt wire reference) in a 200-mM sodium phosphate buffer (pH 8.0) containing 3.5 mM Ru(bpy)(3)(2+) (where bpy=2,2'-bipyridyl). Derivatization of IBAN prior to CE-ECL analysis was not needed. Linear correlation (r=0.9992, n=7) between ECL intensity and analyte concentration was obtained in the range of 0.25-50 µM IBAN. The LOD of IBAN in water was 0.08 µM. The developed method was applied to the analysis of IBAN in a drug formulation and human urine sample. SPE using magnetic Fe(3)O(4)@Al(2)O(3) nanoparticles as the extraction phase was employed to pretreat the urine sample before CE-ECL analysis. The linear range was 0.2-12.0 µM IBAN in human urine (r=0.9974, n=6). The LOD of IBAN in urine was 0.06 µM. Total analysis time including sample preparation was <1 h.

Determination of zoledronic acid in human urine and blood plasma using liquid chromatography/electrospray mass spectrometry.

A new method for the analysis of 1-hydroxy-2-imidazol-1-yl-phosphonoethyl phosphoric acid (zoledronic acid) in urine and blood samples has been developed. It consists of a derivatisation of the bisphosphonate with trimethylsilyl diazomethane under multiple methylester formation. The formed derivative can, in contrast to the non-derivatised analyte, easily be separated by reversed phase liquid chromatography due to its reduced polarity. Detection is performed by electrospray tandem mass spectrometry. For calibration purposes, a deuterated internal standard has been synthesised in a three-step synthesis starting with d(4)-imidazole. For human urine, the limit of detection (LOD) is 1.2x10(-7) mol/L, limit of quantification (LOQ) is 3.75×10(-7) mol/L in the MRM mode. For human blood plasma, a LOD of 1×10(-7) mol/L and a LOQ of 2.5×10(-7) mol/L were determined. The linear dynamic range comprised 3.5 decades starting at the limit of quantification. The method was successfully applied for the analysis of spiked urine and blood plasma samples as well as samples from two osteoporosis patients.

A sensitive post-column photochemical derivatization/fluorimetric detection system for HPLC determination of bisphosphonates.

A new reversed-phase ion-pair high-performance liquid chromatographic (HPLC) method has been developed for the determination of the following bisphosphonic acids: alendronic acid (ALEN), etidronic acid (ETID), ibandronic acid (IBAN) and risedronic acid (RISE). Separation was achieved on a C(18) column using a mixture of 50 mmol L(-1) borate buffer pH 9.0 containing 0.25 mmol L(-1) tetrabutylammonium chloride and 0.5 mmol L(-1) EDTA and acetonitrile (97:3) as the mobile phase. The sensitive detection of the above bisphosphonic acids was based on their oxidation to orthophosphate by the on-line peroxydisulfate-assisted photolysis followed by post-column reaction with molybdate to yield phosphomolybdate. This subsequently reacted with thiamine to generate thiochrome and, finally, the fluorescence of thiochrome was measured at 440 nm with excitation at 375 nm. The developed method is precise with a mean relative standard deviation of 1.3%, sensitive (with a detection limit at the nmol L(-1) level), accurate, specific, rapid (analysis time approximately 13 min) and inexpensive because to the low cost of the reagents. The assay was applied to the analysis of the four bisphosphonic acids in commercial dosage formulations, in which the excipients did not interfere with the determination. The method was also applied to the determination of etidronate, risedronate and ibandronate in human urine. Sample preparation involves precipitation of the analytes from urine along with endogenous phosphates such as calcium salts by addition of calcium chloride at alkaline pH and dissolution of the precipitate in 0.05 mol L(-1) ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid.

Biodistribution and plasma protein binding of zoledronic acid.

The bisphosphonate zoledronic acid is a potent inhibitor of osteoclast-mediated bone resorption. To investigate drug biodistribution and elimination, (14)C-zoledronic acid was administered intravenously to rats and dogs in single or multiple doses and assessed for its in vitro blood distribution and plasma protein binding in rat, dog, and human. Drug exposure in plasma, bones, and noncalcified tissues was investigated up to 240 days in rats and 96 h in dogs using radiometry after dissection. Drug biodistribution in the rat and within selected bones from dog was assessed by autoradiography. Concentrations of radioactivity showed a rapid decline in plasma and noncalcified tissue but only a slow decline in bone, to approximately 50% of peak at 240 days post dose, whereas the terminal half-lives (50-200 days) were similar in bone and noncalcified tissues, suggesting redistribution of drug from the former rather than prolonged retention in the latter. Uptake was highest in cancellous bone and axial skeleton. At 96 h after dose, the fraction of dose excreted was 36% in rat and 60% in dog; 94 to 96% of the excreted radioactivity was found in urine. Blood/plasma concentration ratios were 0.52 to 0.59, and plasma protein binding of zoledronic acid was moderate to low in all species. The results suggest that a fraction of zoledronic acid is reversibly taken up by the skeleton, the elimination of drug is mainly by renal excretion, and the disposition in blood and noncalcified tissue is governed by extensive uptake into and slow release from bone.

Changes of bone turnover markers and serum PTH after night or morning administration of zoledronic acid in breast cancer patients with bone metastases.

Persistent circadian rhythm of bone turnover in bone metastatic breast cancer suggests greater skeletal retention of bisphosphonates if administered in the night. We assessed differential effects of night vs morning administration of zoledronic acid (ZA) on bone turnover. Forty-four breast cancer patients with bone metastases were randomised to receive intravenous ZA (4 mg) at 1100 or 2300 hours every 28 days for four times. Urinary concentration N-telopeptide of type-I collagen (NTX) and deoxypyridinolines, and serum C-telopeptide of type-I collagen (CTX), bone alkaline phosphatase (ALP), osteocalcin and Parathyroid hormone (PTH) was measured in the morning at baseline and after 4, 7, 14, 28, 56 and 84 days. Urinary ZA concentration was also measured. Zoledronic acid caused significant decreases of NTX and CTX (P<0.001), without any difference in percent changes between night and morning arms. Bone ALP and osteocalcin were also significantly affected by ZA (P=0.001), without any difference between arms. Parathyroid hormone significantly increased in both the arms; PTH increase was lower in the night arm (P=0.001). From the second administration onwards, urinary ZA level was significantly higher in the night arm (P<0.01). Administration of ZA at two opposite phases of the circadian cycle causes similar changes of bone-turnover marker levels, but has differential effects on the level of serum PTH.

Development and application of a high-performance liquid chromatography-mass spectrometry method to determine alendronate in human urine.

Despite the high potential offered by electrospray ionization on highly polar compounds like biphosphonates, few applications have been developed. High-performance liquid chromatography (HPLC) separation methods suitable for such molecules cannot be used in tandem with mass spectrometry (MS) due to high non-volatile salt content; at the same time the sample preparation, in biological fluids, is also a challenging problem. In the past ion-pair chromatography was mainly used in the case of HPLC-MS of biphosphonates, but no application to quantitative pharmacokinetic (PK) studies has been presented. In this study, after preliminary tests with ion-pair chromatography showing a poor sensitivity, a combined derivatization of the amino group and the biphosphonate has been developed and tested in a PK study. Using this analytical approach we were able to fully validate the quantitation of alendronate in the range of 6.667-4860.0 ng/ml in urine (sample volume 2.0 ml); each analytical run was 5.0 min long. The sensitivity achieved permitted a correct evaluation of the alendronate urinary excretion over the full period of urine collection. Sample preparation despite its complexity permitted to process and analyze up to 200 samples in a working day.

Evaluation of multidimensional capillary electrophoretic methodologies for determination of amino bisphosphonate pharmaceuticals.

Alendronate and pamidronate are amino bisphosphonate analogues of pyrophosphate used for the treatment of a variety of bone diseases. Analysis of these compounds is problematic due to their polar ionic nature, lack of a suitable chromophore and chelation properties and current analytical approaches involve extensive sample preparation and derivatization procedures. The potential of multidimensional capillary electrophoretic methodological approaches, which eliminate sample preparation have been evaluated for the analysis of these compounds both in aqueous and urinary matrices. Capillary isotachophoresis (cITP) was employed as a pre-separation and on-line sample concentration step prior to analytical determination using either cITP or capillary zone electrophoresis (CZE) with conductivity detection. Single cITP, cITP-cITP and cITP-CZE approaches were partially validated with respect to repeatability, recovery and linearity of response for both compounds. The increases in sensitivity achievable through increasing injection volume from 30 to 300 microL may render such strategies appropriate for determination of these agents at biologically relevant concentrations with minimal sample work-up.

Skeletal retention of bisphosphonate (pamidronate) and its relation to the rate of bone resorption in patients with breast cancer and bone metastases.

UNLABELLED: Bisphosphonate pharmacokinetics may affect individual responses. Skeletal retention of pamidronate infused monthly to patients with bone metastases was highly variable (12-98%) and did not diminish with time, showing the capacity of the skeleton to retain large amounts of bisphosphonate. Relationships between skeletal retention of pamidronate and rate of bone resorption are complex and depend on previous treatment and the total amount of retained bisphosphonate. INTRODUCTION: Bisphosphonates (BPs) given intravenously every 3-4 weeks are effective in the management of metastatic bone disease from breast cancer, but responses among patients vary, and it is not known whether current dose and dose intervals are appropriate for an individual patient. An influence of pharmacokinetics of BPs on antiresorptive action may contribute to this variation in response. To test this hypothesis, we determined the skeletal retention of intravenous pamidronate and its association to the rate of bone resorption in patients with bone metastases from breast cancer. MATERIALS AND METHODS: In a cross-sectional study, 24-h urinary excretion of pamidronate and the biochemical marker of bone resorption N-terminal telopeptide of type 1 collagen and serum alkaline phosphatase were measured in 40 patients with bone metastases from breast cancer at the beginning, after 3-6 months, and after 1 year of treatment with intravenous pamidronate 90 mg every 3-4 weeks. RESULTS AND CONCLUSIONS: Skeletal retention (dose--amount excreted into urine) 2 days after infusion varied between 12% and 98% (mean, 62%) of the administered dose, but there were no differences in retention between patients receiving pamidronate for the first time or after 3-6 months or after 1 year of treatment. Retention of pamidronate was related to the prevalent rate of bone turnover in previously untreated patients, whereas no such relationship was found in previously treated patients. Rate of bone resorption after treatment seemed to be related to the amount of pamidronate retained. During 1 year of treatment, retention of pamidronate remained constant, indicating no saturation of skeletal binding sites with treatment. The variability in retention among individual patients can be attributed to the number of available binding sites. This is related, however, to bone turnover only before the start of treatment. The apparent relationships between skeletal retention and antiresorptive effect could have implications for the design of optimal therapeutic regimens with BPs in patients with bone metastases from breast cancer.

Analytical methods for the quantification of ibandronate in body fluids and bone.

The accurate determination of bisphosphonate levels in bone and biological fluids is important in both clinical and pharmacological/toxicological studies. Ibandronate is a potent nitrogen-containing bisphosphonate containing a tertiary amine group, which does not easily form chromophore derivatives that can be detected by UV light or fluorescence emissions. The current report describes the methodology and validation of a GC-MS assay for ibandronate in serum/plasma and urine, a similar, modified GC-MS method for measurement of bone ibandronate levels, and an ELISA for ibandronate determination in serum/plasma. The range of quantification for the GC-MS was 1-100 ng/ml and 2-7500 ng/ml in plasma or serum and urine, respectively, and 50-1600 pg/ml (potentially 10-320 pg/ml depending on sample size) for the ELISA in plasma or serum. These assays were comparable. The practical application of the assays in preclinical and clinical studies is briefly reviewed.

Short term whole body retention in relation to rate of bone resorption and cartilage degradation after intravenous bisphosphonate (pamidronate) in rheumatoid arthritis.

OBJECTIVE: Bisphosphonates (BP) inhibit osteoclast-mediated bone resorption, and have been reported to decrease the rate of cartilage degradation. The anti-resorptive effect of BP is determined by the amount of BP retained by the skeleton. In rheumatoid arthritis (RA) the uptake is not confined only to the skeleton, but BP is also retained in joints, which could have implications for dose regimens. We investigated the whole body retention (WBR) of pamidronate and its relationship to bone resorption and cartilage degradation in patients with active RA. METHODS: Twenty-six patients received placebo, 45 mg, or 90 mg intravenous pamidronate. Serum and urine samples were collected before and for 12 days after drug administration. Rate of bone resorption was assessed by the biochemical markers: serum carboxy terminal cross-linked telopeptide of type I collagen, urinary carboxy terminal cross-linked telopeptide of type I collagen normalized to creatinine and urinary amino-terminal telopeptide of type I collagen normalized to creatinine; and rate of cartilage degradation by urinary carboxy terminal telopeptide of type II collagen normalized to creatinine. WBR was derived from urinary excretion of pamidronate data. RESULTS: Pamidronate induced a rapid and sustained decrease in the level of biochemical markers of bone resorption and cartilage degradation. The mean WBR of pamidronate was 69% of the administered dose, and showed a remarkably wide range (41-96%). The decrease in rate of bone resorption, but also rate of cartilage degradation appeared to be related to the WBR of pamidronate. CONCLUSION: This is the first study in which the effect of BP treatment has been studied in relation to the amount of BP retained by the body in patients with active RA. The total amount of BP retained by the body shows a remarkably wide range and is comparable with literature on patients with osteoporosis. The apparent relationships between the amount of BP retained by the body and the effect could have implications for therapeutic regimens in patients with RA.

Determination of pamidronate in human whole blood and urine by reversed-phase HPLC with fluorescence detection.

Pamidronate is a bisphosphonate that is effective in treating bone disease including osteopenia and osteoporosis in adults. A sensitive and reliable method for the analysis of pamidronate in whole blood and urine is key to the development of this drug for use in children. A previously described method for pamidronate analysis serum and urine did not consistently detect the drug at satisfactory levels in whole blood. The procedure involves co-precipitation of the bisphosphonates with calcium phosphate, pre-column derivitization with fluorescamine, HPLC utilizing a Nucleosil C(18) column, and fluorescence detection with excitation at 395 nm and emission at 480 nm. Changes to the original protocol included the use of a new internal standard (alendronate), the optimization of the concentration of ethylenediaminetetraacetic acid (EDTA) for dissolving the precipitate, and the elimination of the acidification step prior to deproteinization. The optimum EDTA concentration, which had a significant effect on the labeling capability of fluorescamine, was determined to be 20 mm.A good separation between pamidronate and alendronate was achieved using a heated (40 degrees C ) Nucleosil C(18), 10 micro m particle size column. The mobile phase was an aqueous solution of 1 mm Na(2)EDTA-methanol (97:3, v/v) adjusted to pH 6.5 using a fl ow-rate of 1 mL/min. Fluorescence detection was set at 395 nm for excitation and at 480 nm for emission. The limit of quantitation for pamidronate was 0.5 micro g/mL in whole blood and 0.1 micro g/mL in urine. The method was applied to both whole blood and urine samples from pediatric patients.

Development and validation of a highly sensitive RIA for zoledronic acid, a new potent heterocyclic bisphosphonate, in human serum, plasma and urine.

Zoledronic acid is a new, highly potent bisphosphonate drug under clinical evaluation. A radioimmunoassay has been developed to determine zoledronic acid concentration in human serum, plasma, and urine. The assay utilizes rabbit polyclonal antisera against a zoledronic acid-BSA conjugate and a [125I]zoledronic acid derivative as tracer in a competitive format adapted to microtiter plates. The assay shows a LLOQ 0.4 ng/ml in serum or plasma (interassay%CV=17%, accuracy 97%), 5 ng/ml in urine (21%, 98%). In 23 patients receiving 4, 8 or 16 mg of zoledronic acid, drug concentrations in plasma were dose proportional and showed a multiphasic profile, followed by a prolonged gradual decline to concentrations near the LLOQ. Zoledronic acid disposition in plasma and the recovery of only 40-50% of the dose in urine are consistent with the rapid and extensive uptake by and slow release from bone in parallel with renal clearance, typically shown by bisphosphonates.

New bone-seeking agent: animal study of Tc-99m-incadronate.

OBJECTIVE: Disodium cycloheptylaminomethylenediphosphonate monohydrate (incadronate disodium) is a third-generation bisphosphonate compound which potently inhibits bone resorption, and a highly effective drug in the treatment of metastatic bone disease. We first labeled incadronate disodium with 99mTc, and examined its biodistribution and bone uptake after intravenous injection in rats to assess its potential for clinical use as a bone-seeking agent for judgment of the therapeutic effect of incadronate on bone metastases. Bone scan with 99mTc-labeled incadronate (99mTc-incadronate) may yield important information prior to the use of incadronate for treatment of bone metastases. METHODS: Synthesis of 99mTc-incadronate was carried out by reduction of 99mTc-pertechnetate in the presence of SnCl2 and N2 gas. Normal rats were injected with 18.5 MBq (0.5 mCi) 99mTc-incadronate in a volume of 0.1 ml intravenously and then sacrificed at 15 min, 30 min, 1 h or 2 h (six rats at each time point) after injection. Samples of muscle, stomach, small intestine, kidney, liver and bone (femur) were taken and weighed. In addition, a 1-ml sample of blood was drawn from the heart, and urine was taken from the urinary bladder immediately after sacrifice. Samples were measured for radioactivity and expressed as percent uptake of injected dose per gram or per milliliter (% ID/g or ml). Bone-to-blood and bone-to-muscle uptake ratios were determined from the % ID/g or ml values for these organs. RESULTS: The greatest accumulation of 99mTc-incadronate was found in bone. Radioactivity in bone was as high as 3.22 +/- 0.68% ID/g at 2 hours after injection. Scintigraphic images of 99mTc-incadronate in normal rats revealed highly selective skeletal uptake. CONCLUSION: 99mTc-incadronate exhibited high uptake in bone, and relatively low uptake in soft tissue, suggesting that it may be useful as a bone-seeking agent for judgment of the therapeutic effect of incadronate on bone metastases, by determining the degree of its accumulation in metastatic bone lesions.

[Laboratory diagnosis of osteoporosis]. / Labordiagnostik der Osteoporose.

Bone is biologically a highly active tissue whose cells are embedded in a complex network of systemically acting hormones and local mediators. The mechanisms of action involved are as yet only partially understood. With the increase in life expectancy and the resultant change of the population's age structure, diseases of the musculoskeletal system and bones have increased in importance. Thus, research is directed to a greater extent toward bone metabolism and the most frequent bone disease, osteoporosis. Until a few decades ago, the diagnosis of a bone disease was based principally on clinical and radiological methods. Laboratory methods only included the measurement of total alkaline phosphatase activity and calcium and phosphate balance. The development and introduction of new biochemical markers of bone metabolism in recent years led to a considerable increase in available laboratory methods. To evaluate the activity of osteoblastic synthesis, alkaline phosphatase and other bone-forming markers with higher tissue specificity such as bone alkaline phosphatase, osteocalcin, and several collagen propeptides are used. Bone degradation (calcium and hydroxyproline were the only markers until several years ago) can now be detected quickly and reliably with many new serological and urinary markers. Pyridinium derivatives and telopeptides as products of the metabolic activity of osteoclasts have been proved efficacious in diagnosis and therapy control.