Bone Mineral Content In Rats Research Articles

Water Treatment by Magnetic Field Increases Bone Mineral Density of Rats

Water treatment using a magnetic field is an attractive but controversial issue with regard to its effects on human health. This study aimed to investigate the effects of water treatment using a magnetic field on the bone mineral density (BMD), bone mineral content (BMC), bone area (BA), bone resistance (BR), blood gas analysis, blood viscosity, and blood biochemical profile of rats. Forty-eight Wistar rats were divided into 2 groups: control (n = 24) and magnetic water-treated (n = 24). Each of these groups was subdivided into 3 groups to evaluate 3 consumption periods (15, 30, and 45 d). The animals were kept in metabolic cages throughout the experiment. A completely randomized design distributed to a 2 × 3 factorial arrangement was used. No significant difference was found in the water intake, dry matter intake, BA, or femoral head resistance between the groups. However, higher anion gap and lower CHCO3 were found in the arterial blood of the magnetic water-treated group. There was significant interaction between the water consumption period and the BR, BMD, and BMC. With 15 d of consumption, there was no difference in the BMC and BR. With 30 d of consumption, the BR (midshaft), BMD, and BMC showed increases; the increases were greater with 45 d of consumption. In adulthood, every month of the animal is approximately equivalent to 2.5 human years. The consumption of water treated by magnetic field for 45 d provided an effective way to improve BMD, BMC and BR in rats.

Moderate zinc supplementation during prolonged steroid therapy exacerbates bone loss in rats.

The present study was conducted to understand the influence of zinc on bone mineral metabolism in prednisolone-treated rats. Disturbance in bone mineral metabolism was induced in rats by subjecting them to prednisolone treatment for a period of 8 weeks. Female rats aged 6-8 weeks weighing 150 to 200 g were divided into four treatment groups, viz., normal control, prednisolone-treated (40 mg/kg body weight orally, thrice a week), zinc-treated (227 mg/L in drinking water, daily), and combined prednisolone + zinc-treated groups. Parameters such as changes in mineral levels in the bone and serum, bone mineral density (BMD), bone mineral content (BMC), and bone 99m-technetium-labeled methylene diphosphonate ((99m)Tc-MDP) uptake were studied in various treatment groups. Prednisolone treatment caused an appreciable decrease in calcium levels both in the bone and serum and also in bone dry weight, BMC, and BMD in rats. Prednisolone-treated rats when supplemented with zinc showed further reduction in calcium levels, bone dry weight, BMD, and BMC. The study therefore revealed that moderate intake of zinc as a nutritional supplement during steroid therapy could enhance calcium deficiency in the body and accelerate bone loss.

Implications of combined ovariectomy/multi-deficiency diet on rat bone with age-related variation in bone parameters and bone loss at multiple skeletal sites by DEXA.

BackgroundOsteoporosis is a multi-factorial, chronic, skeletal disease highly prevalent in post-menopausal women and is influenced by hormonal and dietary factors. Because animal models are imperative for disease diagnostics, the present study establishes and evaluates enhanced osteoporosis obtained through combined ovariectomy and deficient diet by DEXA (dual-energy X-ray absorptiometry) for a prolonged time period.Material/MethodsSprague-Dawley rats were randomly divided into sham (laparotomized) and OVX-diet (ovariectomized and fed with deficient diet) groups. Different skeletal sites were scanned by DEXA at the following time points: M0 (baseline), M12 (12 months post-surgery), and M14 (14 months post-surgery). Parameters analyzed included BMD (bone mineral density), BMC (bone mineral content), bone area, and fat (%). Regression analysis was performed to determine the interrelationships between BMC, BMD, and bone area from M0 to M14.ResultsBMD and BMC were significantly lower in OVX-diet rats at M12 and M14 compared to sham rats. The Z-scores were below −5 in OVX-diet rats at M12, but still decreased at M14 in OVX-diet rats. Bone area and percent fat were significantly lower in OVX-diet rats at M14 compared to sham rats. The regression coefficients for BMD vs. bone area, BMC vs. bone area, and BMC vs. BMD of OVX-diet rats increased with time. This is explained by differential percent change in BMD, BMC, and bone area with respect to time and disease progression.ConclusionsCombined ovariectomy and deficient diet in rats caused significant reduction of BMD, BMC, and bone area, with nearly 40% bone loss after 14 months, indicating the development of severe osteoporosis. An increasing regression coefficient of BMD vs. bone area with disease progression emphasizes bone area as an important parameter, along with BMD and BMC, for prediction of fracture risk.

Short Term Effects on Bone Quality Associated with Consumption of Soy Protein Isolate and Other Dietary Protein Sources in Rapidly Growing Female Rats

Beneficial effects of soy protein consumption on bone quality have been reported. The effects of other dietary protein sources such as whey protein hydrolysate (WPH) and rice protein isolate (RPI) on bone growth have been less well examined. The current study compared effects of feeding soy protein isolate (SPI), WPH and RPI for 14 d on tibial bone mineral density (BMD) and bone mineral content (BMC) in intact and ovariectomized (OVX) rapidly growing female rats relative to animals fed casein (CAS). The effects of estrogenic status on responses to SPI were also explored. Tibial peripheral quantitative computerized tomography (pQCT) showed all three protein sources had positive effects on either BMD or BMC relative to CAS (P < 0.05), but SPI had greater effects in both intact and OVX female rats. SPI and E2 had positive effects on BMD and BMC in OVX rats (P < 0.05). However, trabecular BMD was lower in a SPI + E2 group compared to a CAS + E2 group. In OVX rats, SPI increased serum bone formation markers, and serum from SPI-fed rats stimulated osteoblastogenesis in ex vivo. SPI also suppressed the bone resorption marker RatLaps (P < 0.05). Both SPI and E2 increased alkaline phosphatase gene expression in bone, but only SPI decreased receptor activator of nuclear factor-kappaB ligand (RANKL) and estrogen receptor gene expression (P < 0.05). These data suggest beneficial bone effects of a soy diet in rapidly growing animals and the potential for early soy consumption to increase peak bone mass.

Effects of probiotic, prebiotic and synbiotic follow‐up infant formulas on large intestine morphology and bone mineralisation in rats

AbstractThe effects of powder functional follow‐up infant formulas on the morphology of the large intestine and the bone mineral content in rats were investigated. Infant formulas were supplemented with probiotics (Bifidobacterium bifidum and Bifidobacterium longum), prebiotics (galacto‐oligosaccharides (4′‐GOS) at 12, 50 and 100 g kg−1) or synbiotics (bifidobacteria and 4′‐GOS) and administered to seven groups of weanling rats over 30 days. After sacrificing the animals, the caecum and colon were removed and the luminal content pH and the crypt depth and cell density within the epithelial layer were measured. Results showed that groups fed synbiotic diets had significantly (P &lt; 0.05) lower caecal and colonic content pH values compared with the control group. However, no test diet significantly modified the crypt depth or cell density in the caecum. In contrast, all seven test diets significantly increased (P &lt; 0.05) the crypt depth in both the proximal and distal portions of the colon. There was a high correlation (Pearson correlation coefficient 0.870) between caecal and colonic content pH values. On the other hand, colonic content pH was negatively correlated with crypt depth in both the proximal and distal colon and with cell density in the distal colon. The authors previously reported increases in calcium, magnesium and phosphorus absorption using the same infant formulas. In the present study it was concluded that the increase in calcium absorption took place in the distal colon whereas magnesium absorption increased in both the proximal and distal colon, and their contents (especially that of calcium) increased in both the femur and tibia. Copyright © 2007 Society of Chemical Industry

Partial Prevention of Long-Term Femoral Bone Loss in Aged Ovariectomized Rats Supplemented with Choline-Stabilized Orthosilicic Acid

Silicon (Si) deficiency in animals results in bone defects. Choline-stabilized orthosilicic acid (ch-OSA) was found to have a high bioavailability compared to other Si supplements. The effect of ch-OSA supplementation was investigated on bone loss in aged ovariectomized (OVX) rats. Female Wistar rats (n = 58, age 9 months) were randomized in three groups. One group was sham-operated (sham, n = 21), and bilateral OVX was performed in the other two groups. OVX rats were supplemented orally with ch-OSA over 30 weeks (OVX1, n = 20; 1 mg Si/kg body weight daily) or used as controls (OVX0, n = 17). The serum Si concentration and the 24-hour urinary Si excretion of supplemented OVX rats was significantly higher compared to sham and OVX controls. Supplementation with ch-OSA significantly but partially reversed the decrease in Ca excretion, which was observed after OVX. The increase in bone turnover in OVX rats tended to be reduced by ch-OSA supplementation. ch-OSA supplementation increased significantly the femoral bone mineral content (BMC) in the distal region and total femoral BMC in OVX rats, whereas lumbar BMC was marginally increased. Femoral BMD was significantly increased at two sites in the distal region in OVX rats supplemented with ch-OSA compared to OVX controls. Total lumbar bone mineral density was marginally increased by ch-OSA supplementation. In conclusion, ch-OSA supplementation partially prevents femoral bone loss in the aged OVX rat model.

Dietary ratio of n-6/n-3 PUFAs and docosahexaenoic acid: actions on bone mineral and serum biomarkers in ovariectomized rats

Hypoestrogenic states escalate bone loss in animals and humans. This study evaluated the effects of the amount and ratio of dietary n-6 and n-3 polyunsaturated fatty acids (PUFAs) on bone mineral in 3-month-old sexually mature ovariectomized (OVX) Sprague–Dawley rats. For 12 weeks, the rats were fed either a high-PUFA (HP) or a low-PUFA (LP) diet with a ratio of n-6/n-3 PUFAs of 5:1 (HP5 and LP5) or 10:1 (HP10 and LP10). All diets (modified AIN-93G) provided 110.4 g/kg of fat from safflower oil and/or high-oleate safflower oil blended with n-3 PUFAs (DHASCO oil) as a source of docosahexaenoic acid (DHA). Fatty acid analyses confirmed that the dietary ratio of 5:1 significantly elevated the amount of DHA in the periosteum, marrow and cortical and trabecular bones of the femur. Dual-energy X-ray absorptiometry measurements for femur and tibia bone mineral content (BMC) and bone mineral density showed that the DHA-rich diets (HP5 and LP5) resulted in a significantly lower bone loss among the OVX rats at 12 weeks. Rats fed the LP diets displayed the lowest overall serum concentrations of the bone resorption biomarkers pyridinoline (Pyd) and deoxypyridinoline, whereas the bone formation marker osteocalcin was lowest in the HP groups. Regardless of the dietary PUFA content, DHA in the 5:1 diets (HP5 and LP5) preserved rat femur BMC in the absence of estrogen. This study indicates that the dietary ratio of n-6/n-3 PUFAs (LP5 and HP5) and bone tissue concentration of total long-chain n-3 PUFAs (DHA) minimize femur bone loss as evidenced by a higher BMC in OVX rats. These findings show that dietary DHA lowers the ratio of 18:2n-6 (linoleic acid)/n-3 in bone compartments and that this ratio in tissue correlates with reduced Pyd but higher bone alkaline phosphatase activity and BMC values that favor bone conservation in OVX rats.

The effect of a simultaneous dietary administration of xylitol and ethanol on bone resorption

Our previous studies have shown that dietary xylitol supplementation diminishes bone resorption in rats, as well as protects against ovariectomy-induced increase of bone resorption during experimental osteoporosis. Interestingly, ethanol, when given simultaneously with xylitol, is known to increase blood concentration of xylitol. On the other hand, ethanol, when given alone, has been shown to increase bone resorption. The aim of the present study was to evaluate the effects of a simultaneous dietary administration of 10% xylitol and 10% ethanol on bone resorption. Bone resorption was determined using measurement of urinary excretion of hydrogen 3 ( 3H) radioactivity in 3H-tetracycline prelabeled rats. Already 4 days after the beginning of dietary supplementations, excretion of 3H was about 15% lower in the xylitol group (diet supplemented with 10% xylitol) and about 25% lower in the xylitol-ethanol group (diet supplemented with 10% xylitol and 10% ethanol) as compared to the controls. The excretion of 3H in these groups remained smaller than that of the controls throughout the entire study period of 40 days. The excretion of 3H in the xylitol-ethanol group remained also smaller than that of the xylitol group. Bone mineral density and bone mineral content were determined with a peripheral quantitative computed tomography (pQCT) system from the rat tibiae at the end of the experiment. Trabecular bone mineral density and trabecular bone mineral content were significantly greater in the xylitol group and in the xylitol-ethanol group compared to the controls. They were also greater in the xylitol-ethanol group as compared to the xylitol group. Cortical bone mineral density and cortical bone mineral content did not differ significantly between the groups. In conclusion, a simultaneous dietary supplementation with 10% xylitol and 10% ethanol seems to diminish bone resorption and to increase trabecular bone mineral density and trabecular bone mineral content in rats. These effects seem to be stronger than the effects induced by 10% xylitol supplementation alone.

Mandible bone loss in osteoporosis rats.

Change in the mandible during the development of osteoporosis has not been studied extensively. Thus, the present study was undertaken to clarify the target loci in the mandible during the development of experimental osteoporosis in aged female rats. Experimental osteoporosis was studied in 76 Wistar strain female rats, 35 weeks old, by means of ovariectomy and dietary calcium deficiency. The rats were divided into the following three groups: (1) group 1, unoperated basal control (Basal), maintained on a diet containing 1.0% calcium; (2) group 2, sham-operated (Sham), maintained on a diet containing 0.01% calcium; and (3) group 3, ovariecomized (OVX), maintained on a diet containing 0.01% calcium. Fifteen rats of each group, except the basal rats (10 and 6 rats), were killed at 3 and 6 months following ovariectomy, respectively. The mandible was extracted, cleaned, and then subjected to bone mineral content (BMC) and bone mineral density (BMD) analyses using a peripheral quantitative computed tomography (pQCT) bone scanner. Each mandible was scanned from the mesial margin of the first molar to distal margin of the second molar. The results showed that dietary calcium deficiency and ovariectomy caused significant decreases of trabecular and cortical BMCs and BMDs in Sham and OVX rats compared with Basal rats. Decreases due to dietary calcium deficiency were greater than those caused by ovariectomy. However, significant age-related changes were not observed in BMC and BMD in Sham and OVX rats for 3-6 months. In addition, the cortical and trabecular BMCs and BMDs obtained were found to relate to location in the mandible for each group.

L-arginine prevents bone loss and bone collagen breakdown in cyclosporin A-treated rats.

Cyclosporin A is implicated in the pathogenesis of post-transplantation bone disease. Because of recent evidence that cyclosporin A may cause renal and cardiovascular toxicity by inhibiting nitric oxide (NO) activity, and that NO slows bone remodeling and bone loss in animal and human studies, we investigated a possible link between NO production and beneficial effects on bone health in cyclosporin A-treated rats. Thirty-six 10-week-old male rats were assigned to six groups of six animals each, and treated for 4 weeks with: vehicle; cyclosporin A; l-arginine; N G-nitro- l-arginine methylester ( l-NAME, a general inhibitor of NO synthase activity); a combination of cyclosporin A+ l-arginine; and a combination of cyclosporin A+ l-NAME. Whole body and regional (spine and pelvis) bone mineral content of rats were measured under basal conditions and at the end of the treatment period by dual-energy X-ray absorptiometry (DXA) scanning. Femur weights and serum concentrations of pyridinoline, a reliable marker of bone resorption, were measured at the end of the study period. Cyclosporin A-, l-NAME-, and cyclosporin A+ l-NAME-treated rats had significantly lower bone mineral content and femur weights, and significantly higher pyridinoline levels than did control animals. The administration of l-arginine appeared to prevent bone loss caused by cyclosporin A, suggesting that this amino acid, which can be converted to produce NO, might prove useful in preventing disturbed bone modeling and inhibition of bone growth associated with cyclosporin A therapy.

Dissociation of Bone Formation from Resorption during 2-Week Treatment with Human Parathyroid Hormone-Related Peptide-(1–36) in Humans: Potential as an Anabolic Therapy for Osteoporosis1

PTH administration increases bone mass in rodents and in humans. PTH-related protein (PTHrP) binds to and signals via the skeletal PTH receptor. Administration of PTHrP on a once daily basis increases bone mineral content in rats. In humans, PTHrP-(1-36) is equipotent to PTH-(1-34) and is active when administered s.c. These findings suggest that PTHrP might have therapeutic benefit in the treatment of osteoporosis. In this study, 13 postmenopausal estrogen-deficient women received a single daily s.c. dose of PTHrP-(1-36) for a 14-day period to determine whether PTHrP-(1-36) 1) could be given in doses that do not alter systemic mineral homeostasis, but increase markers of bone turnover; and 2) is tolerated without adverse effects. Daily s.c. PTHrP-(1-36) administration caused no significant changes in serum calcium or phosphorus concentrations, fractional calcium excretion, the tubular maximum for phosphorus, fractional calcium excretion, or plasma 1,25-dihydroxyvitamin D concentrations. Nephrogenous cAMP and endogenous PTH-(1-84) declined. Importantly, markers of bone formation trended upward, as reported in subjects treated with PTH. In marked contrast to findings in PTH-treated subjects, in PTHrP-treated subjects, markers of bone resorption declined in a highly significant fashion. These observations indicate that PTHrP-(1-36) treatment uncouples bone formation from resorption, in favor of formation. This uncoupling, if it were to continue over the longer term, would predict that PTHrP-(1-36) might be a potent anabolic therapeutic agent for osteoporosis.

Chronic intramedullary infusion of interleukin-1? increases bone mineral content in rats

Interleukin-1 (IL-1) is known to have a potent bone-resorbing activity in vitro, however, results from in vivo studies are conflicting. We performed experiments with the continuous infusion of recombinant IL-1 alpha directly into the femoral bone marrow of rats for 2 weeks and examined its effects on bone by soft X-ray photographs, bone mineral assessment, and histological examination. Infusion of IL-1 alpha at doses greater than 1 microgram/ml (0.6 ng/hour) caused sclerosis around the infusion site on soft X-ray photographs, and the bone mineral content of IL-1 alpha infused femora was increased significantly. Histologically, extensive periosteal bone formation was observed around the infusion site and small mononuclear cells replaced the normal fat tissue. Infusion of prostaglandin E2 alone produced intramedullary bone formation more extensively. Simultaneous infusion of IL-1 alpha and indomethacin (10(-3) M; 179 ng/hour) inhibited the increase of bone mineral content (BMC) induced by IL-1 alpha. Thus, the chronic intramedullary administration of IL-1 alpha stimulates bone formation, especially in the periosteum, and increases BMC in intact rats.

The effect of high sodium intake on bone mineral content in rats fed a normal calcium or a low calcium diet.

The effect of high sodium intake on bone mineral content of rats fed a normal (0.6% Ca) or a low (0.02% Ca) calcium diet was studied. Rats on a normal calcium diet given 1.8% sodium chloride to drink showed persistent and significant hypercalciuria and subnormal bone mineral content. Total calcium content of femur was significantly lower after 4 months (p < 0.02) and 12 months (p < 0.001). In rats maintained on a low calcium diet (0.02% Ca), a high sodium diet for 8 weeks caused a significant loss of calcium in bone similar to that seen in animals fed a normal calcium diet for 4 months. We conclude that high sodium intake reduces bone mineral content, especially if the diet is low in calcium.

Effects of voluntary exercise on bone mineral content in rats

We used a voluntary running model to explore the relationship between average daily running distance and bone mineral status of rats. A total of 60 male Sprague-Dawley rats were randomly assigned at 6 weeks of age to a sedentary control group (n = 22) or to a group with unlimited access to a running wheel (n = 38). The running distance of exercising rats was monitored daily, and steady-state running levels ranged from 3.2 to 18.1 km/day. At the end of the experimental period, femora and tibiae were dissected and bone mineral content (BMC, g/cm) and bone mineral density (BMD, g/cm2) were measured by single-photon absorptiometry. Cross-sectional morphometry was examined by taking a transverse section of the femoral middiaphysis. Hindlimb percentage fat was significantly higher in controls than in runners (20.0 +/- 1.2 versus 11.1 +/- 0.6, p less than 0.001), and soleus mass was greater in runners than in controls (371 +/- 8.1 versus 320 +/- 0.8 mg, p less than 0.001). Femoral and tibial lengths, weights, and volumes were significantly higher in runners than in controls (p less than 0.005). BMC and BMD were higher in runners than in controls at all sites apart from the distal femur. Cross-sectional areas at the femoral midshaft were greater in running rats than in sedentary controls (6.26 +/- 0.1 versus 5.45 +/- 0.3 mm2, p less than 0.02), as was the polar moment of inertia (15.6 +/- 0.6 versus 12.7 +/- 0.2 mm4, p less than 0.05). No positive correlation was found between distance run and BMC, BMD, cross-sectional area, or polar moment of inertia.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of ovariectomy and prednisolone on bone mineral content in rats: Evaluation by single photon absorptiometry and radiogrammetry

The effects of ovariectomy and prednisolone were studied for 9 months in 3-month-old rats on a moderately low calcium diet. Measurements were made by single photon absorptiometry of the femur at cortical and trabecular bone sites. Radiogrammetry was performed at the midshaft of the femur. During growth, body weight, bone size, and bone mineral content (BMC) increased in male rats more than in females. After ovariectomy, body weight increase was more pronounced, but bone mineral increase was lower than in controls. At the distal end of the femur, bone mineral density decreased after 3 months, and at the midshaft of the femur, medullary width increased significantly from 6 months on. Prednisolone in a dose of 0.5 mg/kg/day did not influence BMC. However, prednisolone treatment after ovariectomy induced a more pronounced effect on bone than ovariectomy alone. BMC increase was lower than in ovariectomy, and bone mineral density decreased significantly, especially at the distal end of the femur. After correction for differences in body weight, globally the same results were found. We conclude that (1) the combination of single photon absorptiometry and radiogrammetry allows the evaluation of growth and the effect of ovariectomy and prednisolone treatment in rats; (2) ovariectomy results in bone loss first at the distal end and later in the midshaft of the femur; (3) prednisolone in a dose of 0.5 mg/kg/day alone did not affect bone mass; and (4) prednisolone profoundly enhanced the effects of ovariectomy.

Nondestructive measurement of bone mineral in femurs from ovariectomized rats

One hundred ninety-eight rats were ovariectomized (OX) or sham-ovariectomized (shOX) at 100 days of age. Groups were killed at 35, 70, 100, 125, 180, 270, 360, and 540 days postsurgery. Bone mineral content (BMC) of the right femur was assayed on a dual photon absorptiometer (DPA) optimized for human spine and whole body measurements. Three regions were studied: the distal measuring 0.8 cm, the proximal measuring 0.88 cm, and the diaphysis, the remainder. The DPA technique accurately showed the ash content (r = 0.96), with a precision error of 3-5%. Whole femoral BMC was 4.3-11.1% lower in OX than shOX rats, with significant differences from 35-180 days. By 35 days, distal femoral BMC declined 6% in OX rats and rose 12% in shOX rats. Distal femoral BMC was 11.3-17.5% lower in OX than shOX rats, with significant differences at all times except 540 days. Femoral diaphyseal BMC of OX and shOX rats did not differ at any time. The relative distal femoral osteopenia which appeared by 35 days in OX rats did not worsen during the next 17 months. A DPA suited for human BMC studies is also accurate for BMC determination in bones with 250-500 mg of mineral. It is less precise for this purpose than dedicated instruments using single photon absorptiometry. However, enough precision exists to monitor the development of relative osteopenia in OX rats. Osteopenia in OX rats is confined to a region containing appreciable cancellous bone. Its self-limiting nature suggests the existence of an estrogen-dependent quantum of cancellous bone in female rats. The adult rat model is accurate for cancellous bone of the adult human, but inaccurate for cortical bone.

In vivo bone mineral analysis throughout skeletal growth in rats: differences due to sex or vitamin D deficiency.

This study reports a new technique for the measurement of bone mineral content (BMC) in live rats. A single photon absorptiometric instrument has been adapted for rapid, reproducible measurement of BMC. Four bone sites were selected for use, based on ease of positioning and reproducibility of measurement; these were as follows: proximal femur, midfemur, proximal seventh caudal vertebra, and midseventh caudal vertebra. Significant increases were detected when BMC was measured at biweekly intervals from weaning to 18 weeks of age in normal male rats. Comparison of body growth and changes in BMC of male and female rats with age showed that body weight gain of female rats slowed earlier than that of male rats whereas BMC increased at similar rates in both sexes. Vitamin D deprivation from day 24 of life resulted in decreased BMC at all four measurement sites compared with such measurements in normal control rats. Differences were detectable after 8 weeks of age and occurred despite the maintenance of serum calcium levels within normal range and only slight reduction in body weights of vitamin D deprived rats. These studies demonstrate that single photon absorptiometry can be used to monitor changes in BMC in live rats on a routine basis without harm to the animals. Changes in BMC such as those due to growth or vitamin D deprivation can easily be quantitated using this technique.