Measurements of fluoride in bone biopsies by neutron activation analysis for the clinical study of osteoporosis

A procedure has been developed to measure fluoride concentration in bone biopsies by neutron activation analysis /NAA/. The NAA procedure is non-destructive so that the bone biopsies can be used subsequently for histological evaluation. The fluoride content is expressed as F/Ca ratio in the bone samples. The fluoride and calcium are measured using the reactions:¹⁹F/n, /²⁰F /t=11.2 s/ and⁴⁸Ca/n,/⁴⁹Ca/t=8.8 m/, respectively. The F/Ca ratio normalizes the fluoride to bone mineral avoiding the use of bone weight which is unreliable with fresh biopsy samples. This ratio also corrects for variations in neutron flux and gamma counting efficiencies. Results by this procedure were compared to biochemical determinations using an ion-selective electrode for fluoride and atomic absorption for calcium. The two methods gave results which agreed within ±5% which is the precision of the NNA procedure. The NAA method provides a simple and non-destructive procedure for fluoride measurement in bone biopsies for clinical studies. The method is now routinely used in our clinical studies for the fluoride measurements on biopsies from osteoporotic patients treated with fluoride therapy for nearly four years.     

The in vivo neutron activation analysis of calcium in the skeleton of normal subjects,with hypokinesia and bone diseases

A facility developed for the in-vivo neutron activation analysis (IVNAA) of calcium in some parts of tubular limb bones, in the hand, foot and spine is described. The⁴⁸Ca(n, )⁴⁹Ca reaction is used, the gamma-rays of⁴⁹Ca (E=3.08 MeV, T_1/2=8.8 min) are measured. The irradiation is carried out with five of ten²³⁸Pu–Be neutron sources with a source stength of 5·10⁷ n s^–1 each arranged in a bath filled with water. NaI(T1) detectors (from 2 to 4 in number) of 150×100 mm size supplied with lead and shadow shielding are used in the counting unit. The construction of the facility started in 1972 and has been in operation since 1974. It has been used for the examination of patients suffering from limb bone tumors, of children with rickets-like and similar diseases and for studies in space medicine.     

In vivo measurement of the Ca/P ratio by local activation with isotopic neutron sources

In connection with the study and the treatment of generalised demineralising bone diseases, the amount of phosphorus and calcium has been determined in the hand by “in vivo” neutron activation analysis using²⁵²Cf and²³⁸Pu−Be isotopic neutron sources. The statistical accuracy of the induced radioactivity measurement carried out on the hand of a normal subject is about 2% for P and Ca, while the standard deviation over a series of 10 analyses performed on a same phantom remains within 3% for both P and Ca. The results are normalized and expressed in grams of P or Ca per cm³ of bone. The values observed on a group of 55 normal subjects and on some osteoporotic patients are given.     

Non-destructive bone aluminum assay by neutron activation analysis

A non-destructive method based on instrumental neutron activation analysis (INAA) for the assay of aluminum in bone samples is described. The²⁸Al signal obtained upon neutron irradiation includes contributions from both the reaction²⁷Al(n,)²⁸Al and³¹P(n,)²⁸Al. The first reaction is with the thermal neutrons and the second one is with the fast neutrons. The contribution from the³¹P reaction is calculated from the fact that Ca/P ratio in bone mineral is constant and the amount of calcium can be measured from the thermal reaction⁴⁸Ca(n, )⁴⁹Ca. The aluminum values obtained by the INAA procedure agreed within 10% of those obtained by atomic absorption spectrophotometry. With this assay the levels of aluminum in normal bones (<70 g g^–1 apatite) cannot be determined reliably but higher aluminum levles in bone biopsies associated with Al toxicity, e.g. some patients with renal osteodystrophy, can be determined with a precision of ±10%.     

From potential to reality: Yeasts derived from ethanol production for animal nutrition

A chemical and radiochemical neutron activation analysis (CNAA/RNAA) method has been developed for the determination of three calcium isotopes (⁴⁸Ca,⁴⁶Ca, and⁴⁴Ca) in a single sample derived from urine. This method was developed in support of clinical research using a dual enriched stable isotope methodology to study bone mineralization in premature infants, juvenile rheumatoid arthritics, and cystic fibrosis. In these studies, one enriched isotope of calcium is administered orally, and one is administered intravenously. By making determinations of three isotopes (two enriched, one unenriched) within the same sample, the perturbation from natural isotopic ratios can be determined and used to calculate true absorption of calcium. In our method,⁴⁸Ca is determined via the⁴⁸Ca(n,γ)⁴⁹Ca reaction and 3084 keV gamma-ray,⁴⁶Ca via the⁴⁶Ca(n,γ)⁴⁷Ca reaction and 1296 keV gamma-ray, and⁴⁴Ca via the⁴⁴Ca(n,γ)⁴⁵Ca reaction and 256 keV (max) beta-particle. A pair of chemical separation steps are employed to separate calcium from urine as calcium oxalate with a yield in the range of 80–90%, and a radiochemical step is employed prior to the measurement of⁴⁵Ca to remove interfering radionuclides.     

Measurement of calcium and titanium from 47sc by instrumental neutron activation analysis

A procedure is outlined for measuring calcium and titanium from ⁴⁷Sc by instrumental neutron activation analysis. Precise calcium measurements obtained from ⁴⁷Sc are compared with measurements based on ⁴⁷Ca and ⁴⁹Ca. The method is particularly suitable for low levels (ca. 1%) of calcium.     

Determination of calcium,strontium and barium in single crystals of alkali halides by neutron activation

The determination by neutron activation of trace quantities of calcium, strontium and barium in single crystals of alkali halides has been studied. The matrix is eliminated before the irradiation by an ion-exchange procedure. Gamma spectroscopy after radiochemical separation is used to determine strontium and barium. Calcium is determined by measuring the β-activity of ⁴⁹Sc formed by the decay of ⁴⁹Ca. Long-lived radioactive tracers are used to determine the chemical yields.     

Determination of the sodium-to-calcium ratio in sections of undecalcified bone tissue by neutron activation analysis

The feasibility has been shown of instrumental determination of the Na/Ca ratio with a standard deviation of 4.7% in sections of undecalcified bone tissue made from standard biopsy or autopsy bone samples (about 0.5 mg in weight) embedded in poly (methyl methacrylate) blocks. The determination was based on short-time activation and counting the²⁴Na and⁴⁹Ca radionuclides. Blank values and detection limits of the elements were evaluated and possibilities to detect changes of the Na/Ca ratio are discussed.     

Labeling the human skeleton with 41Ca to assess changes in bone calcium metabolism

Bone research is limited by the methods available for detecting changes in bone metabolism. While dual X-ray absorptiometry is rather insensitive, biochemical markers are subject to significant intra-individual variation. In the study presented here, we evaluated the isotopic labeling of bone using ⁴¹Ca, a long-lived radiotracer, as an alternative approach. After successful labeling of the skeleton, changes in the systematics of urinary ⁴¹Ca excretion are expected to directly reflect changes in bone Ca metabolism. A minute amount of ⁴¹Ca (100 nCi) was administered orally to 22 postmenopausal women. Kinetics of tracer excretion were assessed by monitoring changes in urinary ⁴¹Ca/⁴⁰Ca isotope ratios up to 700 days post-dosing using accelerator mass spectrometry and resonance ionization mass spectrometry. Isotopic labeling of the skeleton was evaluated by two different approaches: (i) urinary ⁴¹Ca data were fitted to an established function consisting of an exponential term and a power law term for each individual; (ii) ⁴¹Ca data were analyzed by population pharmacokinetic (NONMEM) analysis to identify a compartmental model that describes urinary ⁴¹Ca tracer kinetics. A linear three-compartment model with a central compartment and two sequential peripheral compartments was found to best fit the ⁴¹Ca data. Fits based on the use of the combined exponential/power law function describing urinary tracer excretion showed substantially higher deviations between predicted and measured values than fits based on the compartmental modeling approach. By establishing the urinary ⁴¹Ca excretion pattern using data points up to day 500 and extrapolating these curves up to day 700, it was found that the calculated ⁴¹Ca/⁴⁰Ca isotope ratios in urine were significantly lower than the observed ⁴¹Ca/⁴⁰Ca isotope ratios for both techniques. Compartmental analysis can overcome this limitation. By identifying relative changes in transfer rates between compartments in response to an intervention, inaccuracies in the underlying model cancel out. Changes in tracer distribution between compartments were modeled based on identified kinetic parameters. While changes in bone formation and resorption can, in principle, be assessed by monitoring urinary ⁴¹Ca excretion over the first few weeks post-dosing, assessment of an intervention effect is more reliable ∼150 days post-dosing when excreted tracer originates mainly from bone.     

Minimum Detectable Mass Fraction of Localised Changes in Biological Matrices Using Neutrons in Transmission

The value of the minimum detectable mass of an irregularity, which will change the neutron macroscopic cross section of a biological matrix by 1%, has been calculated by using neutron transmission technique. The biological matrices selected were ones in which neutron transmission provided better detection of elements than photon attenuation measurements. Best results were obtained, for the detection of bone marrow in bone (12.2 mg/g), Ca in bone (10 mg/g) and water in brain (6 mg/g). The minimum required number of neutrons and the exposure time required for the detection of irregularities in bone marrow have been calculated.     

INAA application in the age dynamics assessment of Ca, Cl, K, Mg, Mn, Na, P and Sr in the cortical bone of human femoral neck

The effect of age and sex on eight elements in cortical bone of femoral neck of 78 relatively healthy 15-55 years old women (n = 33) and men (n = 45) was studied. Mass fraction of Ca, Cl, K, Mg, Mn, Na, P, and Sr in intact bone samples were determined by instrumental neutron activation analysis using short-lived radionuclides. The obtained results were in good agreement with the literature data with the exception of Mn and Sr. The mean value of Mn mass fraction was an order of magnitude lower and Sr mean mass fraction was in 1.5-5 times higher than the reference values. No age- and sex-related differences in bone composition were detected. This revised version was published online in August 2006 with corrections to the Cover Date.     

Small sample in-vivo neutron activation analysis using californium sources

This work describes an in vivo neutron activation analysis facility for small samples, such as rats or human hand, using two 100 g²⁵²Cf neutron sources. The irradiation area is a cylindrical space, of 12 cm diameter and about 15 cm length, with fairly uniform neutron flux distribution. Experimental data on the reproducibility, effects of volume and other conditions for in vivo measurements are given. Comparative atomic absorption data on calcium measurements on rats are reported. The facility is now used for animal experiments as well as human hand irradiations in clinical investigations involving calcium metabolism and bone diseases.     

Evaluation of New Pharmaceuticals Using In Vivo Neutron Inelastic Scattering and Neutron Activation Analysis

Nutritional status of patients can be evaluated by monitoring changes in body composition, including depletion of protein and muscle, adipose tissue distribution and changes in hydration status, bone or cell mass. Fast neutron activation (for N and P) and neutron inelastic scattering (for C and O) are used to assess in vivo elements characteristic of specific body compartments. The fast neutrons are produced with a sealed deuterium-tritium (D-T) neutron generator. This method provides the most direct assessment of body composition. Non-bone phosphorus for muscle is measured by the ³¹P(n,)²⁸Al reaction, and nitrogen for protein via the (n,2n) fast neutron reaction. Inelastic neutron scattering is used for the measurement of total body carbon and oxygen. Carbon is used to derive body fat, after subtracting carbon contributions due to protein, bone and glycogen. Carbon-to-oxygen (C/O) ratio is used to measure distribution of fat and lean tissue in the body and to monitor small changes of lean mass and its quality. In addition to evaluating the efficacy of new treatments, the system is used to study the mechanisms of lean tissue depletion with aging and to investigate methods for preserving function and quality of life in the elderly.     

Calibration strategies for quantifying the Mn content of tooth and bone samples by LA-ICP-MS

There is a growing interest in using biomonitoring of tooth and bone specimens to assess human exposure to manganese (Mn). Information on historical exposure to Mn can be obtained through micro-spatial analysis of such specimens by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The principal aim of this study was to compare several calibration strategies for determining Mn content in tooth and bone by LA-ICP-MS including: (a) a six-point calibration curve based on synthesized hydroxyapatite (HA) materials, and single-point calibrations based on (b) NIST SRM 1400 Bone Ash, (c) NIST SRM 1486 Bone Meal, and (d) NIST SRM 612 Trace Elements in Glass. Performance was similar between different ICP-MS platforms (quadrupole ICP-MS, dynamic reaction cell ICP-MS, and sector field ICP-MS). Data based on calibration using the ⁵⁵Mn count rate were compared to that based on using the ⁵⁵Mn/⁴³Ca count rate ratio to obtain results as the Mn mass fraction. Reasonable performance was obtained by calibration using either SRM 612 or SRM 1400, in combination with the ⁵⁵Mn/⁴³Ca count rate ratio and using either the synthesized HA standards or SRM 1400 as calibrators, combined with ⁵⁵Mn count rate. By contrast, calibration based on SRM 1486 resulted in a systematic low bias. While there are several options for quantifying the Mn content of tooth and bone using LA-ICP-MS, users should be aware of the potential for strong matrix effects that may affect results. Overall, determining the ⁵⁵Mn/⁴³Ca count rate ratio, rather than the mass fraction, may represent a better approach for reporting the content of Mn in tooth and bone by LA-ICP-MS.     

Application of neutron activation analysis to the measurement of isotope abundance shifts in calcium and potassium

A simple method for the measurement by neutron activation analysis of the isotopic abundance shifts of⁴⁴Ca and⁴¹K in compounds of low volatility is described. Extraction and measurement procedures for the radioactive argon isotopes (³⁷Ar,³⁹Ar and⁴¹Ar produced by (n, α) and (n, p) reactions are also given. The results obtained demonstrate that the accuracy for the measurement of δ⁴⁴Ca is better than by the mass spectrometric method; in the case of δ⁴¹K measurement the accuracy is the same as that obtained with the mass spectrometer. Interfering reactions which yield other rare gases, and their relative contribution to the error in the measurement of δ values are also discussed.     

INAA of Ca, Cl, K, Mg, Mn, Na, P, and Sr contents in the human cortical and trabecular bone

Summary Concentration of Ca, Cl, K, Mg, Mn, Na, P, and Sr were determined by instrumental neutron activation analysis using short-lived radionuclides in intact cortical and trabecular bone of femoral neck and iliac crest of 81 relatively healthy 15-55 years old women (n = 36) and men (n = 45). In cortical bone the Ca, P, and Mg mass fractions in the femoral neck were statistically significantly higher, and Cl, K, and Na lower, than the values for the iliac crest. In trabecular bone the Cl, K, and Na mass fractions in the iliac crest were significantly higher, and Ca, P also higher, than the values for the femoral neck.     

Use of D–T-produced fast neutrons for in vivo body composition analysis: a reference method for nutritional assessment in the elderly

Body composition has become the main outcome of many nutritional intervention studies including osteoporosis, malnutrition, obesity, AIDS, and aging. Traditional indirect body composition methods developed with healthy young adults do not apply to the elderly or diseased. Fast neutron activation (for N and P) and neutron inelastic scattering (for C and O) are used to assess in vivo elements characteristic of specific body compartments. Non-bone phosphorus for muscle is measured by the ³¹P(n,)²⁸Al reaction, and nitrogen for protein via the ¹⁴N(n,2n)¹³N fast neutron reaction. Inelastic neutron scattering is used to measure total body carbon and oxygen. Body fat is derived from carbon after correcting for contributions from protein, bone, and glycogen. Carbon-to-oxygen ratio (C/O) is used to measure the distribution of fat and lean tissue in the body and to monitor small changes of lean mass. A sealed, D–T neutron generator is used for the production of fast neutrons. Carbon and oxygen mass and their ratio are measured in vivo at a radiation exposure of less than 0.06 mSv. Gamma-ray spectra are collected using large BGO detectors and analyzed for the 4.43 MeV state of carbon and 6.13 MeV state of oxygen, simultaneously with the irradiation. P and N analysis by delayed fast neutron activation is performed by transferring the patient to a shielded room equipped with an array of NaI(Tl) detectors. A combination of measurements makes possible the assessment of the quality of fat-free mass. The neutron generator system is used to evaluate the efficacy of new treatments, to study mechanisms of lean tissue depletion with aging, and to investigate methods for preserving function and quality of life in the elderly. It is also used as a reference method for the validation of portable instruments of nutritional assessment.     

A search for losses of chemical elements during freeze-drying of biological materials

Possible losses of seven chemical elements were investigated in biological tissues during freeze-drying in vacuum. Thyroid glands were taken during post-mortem examination of 23 people died of different diseases. Instrumental neutron activation analysis (INAA) was used to estimate contents of Br, Ca, Cl, I, K, Mg, and Na. The nuclear reator vertical channel with flux density of 1.2·10¹³n·cm^–2·s^–1 was used for neutron irradiation. The analysis was carried out using short-lived radionuclides induced in samples after neutron irradiation. Then thyroids were freeze-dried at below 0 °C in vaccum up to the constant mass (lyophilisation) and then homogenized. Samples of lyophilised and homogenized tissues were again studied by INAA. The lack of difference between the results of the analysis before and after lyophilisation is an evedence of no loss of Br, Ca, Cl, I, K, Mg and Na during freeze-drying of biotissues in vaccum.     

Detector shielding in the study of osteoporosis by in vivo neutron activation analysis

Calcium may be measured in vivo by neutron activation analysis /IVNAA/, observing the 3.1 MeV -ray from the decay of⁴⁹Ca. Normally the detection of this -ray is done in a heavily shielded whole body counter. It is shown that the use of shielding is unnecessary. As a result, the cost of building an IVNAA facility for Ca is very much reduced.     

Neutron activation analysis of Ca, Cl, Mg, Na, and P content in human bone affected by osteomyelitis or osteogenic sarcoma

The Ca, Cl, Mg, Na, and P content and Ca/P, Ca/Mg, Ca/Na, Cl/Ca, and Cl/Na ratios in samples of intact cortical bone, inflamed bone and osteogenic sarcoma tissue were investigated by neutron activation analysis with high resolution spectrometry of short-lived radionuclides. In osteogenic sarcoma tissue the mass fractions of Cl and Na are higher and the mass fraction of Ca is lower than that of both normal and inflamed bone tissues. It was shown that the differences between the Cl/Ca ratio can be used as an additional test for differential diagnosis between normal or inflamed bone and osteogenic sarcoma.