Assessment of quantitative [18F]Sodium fluoride PET measures of knee subchondral bone perfusion and mineralization in osteoarthritic and healthy subjects.

OBJECTIVE: Molecular information derived from dynamic [18F]sodium fluoride ([18F]NaF) PET imaging holds promise as a quantitative marker of bone metabolism. The objective of this work was to evaluate physiological mechanisms of [18F]NaF uptake in subchondral bone of individuals with and without knee osteoarthritis (OA). METHODS: Eleven healthy volunteers and twenty OA subjects were included. Both knees of all subjects were scanned simultaneously using a 3T hybrid PET/MRI system. MRI MOAKS assessment was performed to score the presence and size of osteophytes, bone marrow lesions, and cartilage lesions. Subchondral bone kinetic parameters of bone perfusion (K1), tracer extraction fraction, and total tracer uptake into bone (Ki) were evaluated using the Hawkins 3-compartment model. Measures were compared between structurally normal-appearing bone regions and those with structural findings. RESULTS: Mean and maximum SUV and kinetic parameters Ki, K1, and extraction fraction were significantly different between Healthy subjects and subjects with OA. Between-group differences in metabolic parameters were observed both in regions where the OA group had degenerative changes as well as in regions that appeared structurally normal. CONCLUSIONS: Results suggest that bone metabolism is altered in OA subjects, including bone regions with and without structural findings, compared to healthy subjects. Kinetic parameters of [18F]NaF uptake in subchondral bone show potential to quantitatively evaluate the role of bone physiology in OA initiation and progression. Objective measures of bone metabolism from [18F]NaF PET imaging can complement assessments of structural abnormalities observed on MRI.

A Prospective Assessment of Periprosthetic Bone Mineral Density and Osteoimmunological Biomarkers Variations After Total Knee Replacement Surgery.

Aseptic loosening is a major cause of premature failure of total knee replacement (TKR). Variations in periprosthetic bone mineral density (BMD) and osteoimmunological biomarkers levels could help to quantify prosthesis osteointegration and predict early aseptic loosening. The gene expression of 5 selected osteoimmunological biomarkers was evaluated in tibial plateau bone biopsies by real-time polymerase chain reaction and changes in their serum levels after TKR were prospectively evaluated with enzyme-linked immunosorbent assay for 1 yr after surgery. These variations were correlated to changes in periprosthetic BMD. Sixteen patients were evaluated. A statistically significant decrease in serum levels of Sclerostin (p = 0.0135) was observed immediately after surgery. A specular pattern was observed between dickkopf-related protein 1 and osteoprotegerin expression. No statistically significant changes were detectable in the other study biomarkers. Periprosthetic BMD did not change significantly across the duration of the follow-up. Prosthetic knee surgery has an impact on bone remodeling, in particular on sclerostin expression. Although not showing statistically significant changes, in the patterns of dickkopf-related protein 1, osteoprotegerin, and the ligand of the receptor activator of nuclear factor kappa-B symmetries and correspondences related to the biological activities of these proteins could be identified. Variation in osteoimmunological biomarkers after TKR surgery can help in quantifying prosthesis osteointegration.

Longitudinal assessment of bone quality in pediatric patients with chronic kidney disease in relation to treatment modality.

Children with chronic kidney disease (CKD) are at high risk of developing impaired bone quality. Our aim was to investigate changes of bone quality in children with CKD in relation to their treatmant using two imaging techniques-dual energy X-ray absorptiometry and quantitative ultraSonography (QUS). Thirty-three patients with CKD (18 boys and 15 girls, mean age 10.37 ± 3.37 years) were evaluated with bone mineral density (BMD) measured by DXA at the lumbar spine and hip and with speed of sound (SOS) measured by QUS at the radius and tibia at the beginning and at the end of the study. The patient cohort consisted of 14 patients with CKD stage 3-4 not treated with dialysis (CKD group), 5 patients on peritoneal dialysis treatment (PD group) and 14 patients after kidney transplantation (RTx group). BMD measurements did not show any significant changes in CKD and PD patients during the study. There was a reduction in BMD measured at the lumbar spine, femoral neck and total hip in RTx patients that was approaching significance. During the 2-year follow-up, SOS measurements at the radius decreased significantly in PD patients, whereas SOS measurements at the tibia significantly improved in RTx patients. No significant changes in QUS parameters were recorded for patients in the CKD group. In conclusion, our study shows that QUS parameters seem to better reflect the state of hyperparathyroidism of renal osteodystrophy as they deteriorate significantly in patients on dialysis and improve after renal transplantation.

Direct assessment of articular cartilage and underlying subchondral bone reveals a progressive gene expression change in human osteoarthritic knees.

OBJECTIVE: To evaluate the interaction of articular cartilage (AC) and subchondral bone (SB) through analysis of osteoarthritis (OA)-related genes of site-matched tissue. DESIGN: We developed a novel method for isolating site-matched overlying AC and underlying SB from three and four regions of interest respectively from the human knee tibial plateau (n = 50). For each site, the severity of cartilage changes of OA were assessed histologically, and the severity of bone abnormalities were assessed by microcomputed tomography. An RNA isolation procedure was optimized that yielded high quality RNA from site-matched AC and SB tibial regions. Quantitative polymerase chain reaction (Q-PCR) analysis was performed to evaluate gene expression of 61 OA-associated genes for correlation with cartilage integrity and bone structure parameters. RESULTS: A total of 27 (44%) genes were coordinately up- or down-regulated in both tissues. The expression levels of 19 genes were statistically significantly correlated with the severity of AC degeneration and changes of SB structure; these included: ADAMTS1, ASPN, BMP6, BMPER, CCL2, CCL8, COL5A1, COL6A3, COL7A1, COL16A1, FRZB, GDF10, MMP3, OGN, OMD, POSTN, PTGES, TNFSF11 and WNT1. CONCLUSIONS: These results provide a strategy for identifying targets whose modification may have the potential to ameliorate pathological alterations and progression of disease in both AC and SB simultaneously. In addition, this is the first study, to our knowledge, to overcome the major difficulties related to isolation of high quality RNA from site-matched joint tissues. We expect this method to facilitate advances in our understanding of the coordinated molecular responses of the whole joint organ.

Small-animal PET/CT assessment of bone microdamage in ovariectomized rats.

UNLABELLED: Microdamage in bone contributes to bone fragility in postmenopausal women. Therefore, it is important to find a noninvasive method to detect microdamage in living bone. PET with (18)F-fluoride has been used for skeletal imaging in clinical studies. However, few studies are undertaken to investigate bone microdamage associated with osteoporosis in vivo using noninvasive means. The aim of our study was to analyze the impact of osteoporosis due to estrogen deficiency on the occurrence of microdamage by observing the change in the uptake of (18)F-fluoride in the tibiae of ovariectomized rats after fatigue loading with small-animal PET/CT. We also explored the feasibility of noninvasive detection of bone microdamage in vivo using a small-animal PET/CT scanner specially designed for rodent study. METHODS: Rats were randomized into 2 groups: ovariectomy and sham surgery. These rats were imaged using a dedicated small-animal PET scanner with (18)F-fluoride after the left tibiae were loaded cyclically under the axial compression. The fluoride uptake values were quantified in the tibial mid shafts, and the tibia was obtained for histomorphometric measurements of bone microdamage and osteocyte density. Bone mineral density at the fourth lumbar vertebra and right femur were measured using dual-energy x-ray absorptiometry. RESULTS: PET image intensity was significantly increased (P < 0.05) in the loaded tibia of the ovariectomy group, compared with that of the sham group. Histomorphometry showed that both crack density and crack length in the loaded tibia were significantly higher (P < 0.05) in ovariectomized rats than in sham rats. The PET image intensity in the loaded tibia was significantly positively correlated with crack length and crack density (which show in histomorphometric measurement) (P < 0.05). CONCLUSION: Both small-animal PET/CT and histomorphometric measurement provided evidence that bone microdamage is significantly increased after estrogen depletion. The strong correlation between these 2 measurements suggests that small-animal PET/CT is a useful noninvasive means to detect bone microdamage in vivo.

Quantitative assessment of the bone morphogenetic protein expression from alternate bone graft harvesting sites.

OBJECTIVE: Bone morphogenetic proteins (BMPs) play important roles in the stimulation of osteogenesis and osteoinduction during bone fracture healing and their expression levels may be important for bone graft efficacy. The objective of this study was to determine if there are variations in the expression of BMPs and their receptors in various bone graft harvesting sites. We analyzed autogenous marrow aspirates obtained from three different graft sites for the mRNA levels of BMPs and their receptors. METHODS: Using real-time polymerase chain reaction, we analyzed the mRNA levels of BMPs and their receptors in autogenous bone marrow aspirates obtained from three different bone graft sites of 10 different human subjects. Collection of autogenous bone marrow from the iliac crest, the proximal humerus, and the proximal tibia was performed using standard sterile techniques in the operating room as part of surgery to treat an established fracture nonunion. RESULTS: The mRNA levels of BMP-2 and BMP-5 were the highest in the bone marrow aspirates from the three different sites, whereas the mRNA levels of the other osteoinductive BMPs (BMP-4, -5, -6, -7, -8, and -9) were lower. The mRNA levels of BMP-3, an inhibitor of osteogenesis, were the lowest in the bone marrow aspirates of all three different sites. There were no statistical significant differences in the mRNA levels of any of the BMPs or their receptors investigated in this study in the bone marrow of the three different sites. CONCLUSION: Because no statistical significant differences in the mRNA levels of the BMPs and their receptors were detected in the bone marrow aspirates from the three different sites, our findings suggest that potential differences of various graft sites in the augmentation of bone healing does not result from different expression levels of BMPs.

Gender and race/ethnicity differences in lead dose biomarkers.

OBJECTIVES: We sought to identify predictors of lead concentrations in the blood, tibias, and patellae of older adults and to describe differences by gender, race/ethnicity, and other factors that can influence lead toxicokinetics and, thus modify health effects. METHODS: Participants aged 50 to 70 years (N=1140) were randomly identified from selected neighborhoods in Baltimore, Maryland. We measured lead concentrations by anodic stripping voltammetry (in blood) and (109)Cd-induced K-shell x-ray fluorescence (in bone). We used multiple linear regression to identify predictors of lead concentrations. RESULTS: Mean (SD) lead concentrations in blood, tibias, and patellae were 3.5 (2.4) mug/dL, 18.9 (12.5) mug/g, and 6.8 (18.1) mug/g, respectively. Tibia concentrations were 29% higher in African Americans than in Whites (P < .01). We observed effect modification by race/ethnicity on the association of gender and physical activity to blood lead concentrations and by gender on the association of age to tibia lead concentrations. Patella lead concentrations differed by gender; apolipoprotein E genotype modified this relation. CONCLUSIONS: African Americans evidenced a prominent disparity in lifetime lead dose. Women may be at higher risk of release of lead from bone and consequent health effects because of increased bone demineralization with aging.

In vivo assessment of trabecular bone microarchitecture by high-resolution peripheral quantitative computed tomography.

CONTEXT: Assessment of trabecular microarchitecture may enhance the prediction of fracture risk and improve monitoring of treatment response. A new high-resolution peripheral quantitative computed tomography (HR-pQCT) system permits in vivo assessment of trabecular architecture and volumetric bone mineral density (BMD) at the distal radius and tibia with a voxel size of 82 microm3. OBJECTIVE AND PATIENTS: We determined the short-term reproducibility of this device by measuring 15 healthy volunteers three times each. We compared HR-pQCT measurements in 108 healthy premenopausal, 113 postmenopausal osteopenic, and 35 postmenopausal osteoporotic women. Furthermore, we compared values in postmenopausal osteopenic women with (n = 35) and without previous fracture history (n = 78). DESIGN AND SETTING: We conducted a cross-sectional study in a private clinical research center. INTERVENTION AND MAIN OUTCOME MEASURE: We took HR-pQCT measurements of the radius and tibia. Femoral neck and spine BMD were measured in postmenopausal women by dual-energy x-ray absorptiometry. RESULTS: Precision of HR-pQCT measurements was 0.7-1.5% for total, trabecular, and cortical densities and 2.5-4.4% for trabecular architecture. Postmenopausal women had lower density, trabecular number, and cortical thickness than premenopausal women (P < 0.001) at both radius and tibia. Osteoporotic women had lower density, cortical thickness, and increased trabecular separation than osteopenic women (P < 0.01) at both sites. Furthermore, although spine and hip BMD were similar, fractured osteopenic women had lower trabecular density and more heterogeneous trabecular distribution (P < 0.02) at the radius compared with unfractured osteopenic women. CONCLUSION: HR-pQCT appears promising to assess bone density and microarchitecture at peripheral sites in terms of reproducibility and ability to detect age- and disease-related changes.

Assessment of experimental osteoporosis using CT-scanning, quantitative X-ray analysis and impact test in calcium deficient ovariectomized rats.

INTRODUCTION: Osteoporosis is a major health problem in the elderly population particularly in postmenopausal women. Various techniques are currently available for detection and diagnosis of osteoporosis in humans, but not all of these can be used for evaluation of osteoporosis in animals. We assessed the feasibility and applicability of a spiral CT-scanning technique for use in evaluating bone loss in rats. In addition, we also tested the applicability of dispersive quantitative X-ray analysis (QXRA) coupled with scanning electron microscopy (SEM), and impact test to detect bone mineral content and biomechanical strength, respectively. METHODS: Twenty female Sprague Dawley rats were randomly assigned to sham operated or bilaterally ovariectomized groups and fed with a calcium-deficient diet for 16 weeks. Femurs and tibias were collected at termination. Using a spiral CT-scanner various cortical bone morphometric indices were measured in femurs. SEM-QXRA was used to quantify the calcium to phosphorus ratio (Ca:P) in regions of high bone turnover both in sham and CD-OVX rats. Tibias were subjected to the impact test for evaluation of biomechanical strength. RESULTS: Compared to sham operated rats, the CD-OVX animals had decreased combined cortical thickness (CCT), cortical area (CA), cortical area/periosteal area and radial density (RD), with increased medullary width and cross sectional areas as determined by CT-Scanning technique. SEM-QXR analysis indicates a decreased Ca:P ratio in the metaphyseal regions of distal femurs in CD-OVX rats when compared to sham-operated rats. The results of the impact test demonstrate lower threshold energy in tibias of CD-OVX rats compared to sham operated rats. DISCUSSION: The ability of these techniques to distinguish the bone characteristics of sham-operated rats from CD-OVX rats provides a methodological opportunity to assess osteoporosis in small animals like rats.

Assessment of changes due to the long-term effect of estrogen and calcium deficiency in the trabecular bone structure in rats.

OBJECTIVE: To assess structural changes, especially structural anisotropy, of rat bone trabecular system 6 months after ovariectomy followed by low-calcium diet. METHOD: The study was carried out on the group of 32 female rats, half of which were ovariectomized at the age of 75 days. The animals were divided into 4 groups: one receiving a normal diet (N), another receiving a low-calcium diet (LCa), ovariectomized rats receiving a normal diet (OVX), and ovariectomized animals receiving a low-calcium diet (OVX+LCa). After 6 months the animals were killed, bone specimens were collected and cut into sections of 6 microm thickness. Digital images of the sections were analyzed using a software package enabling analysis of the transversal and longitudinal trabeculae. RESULTS: Significant changes in trabecular structure due to a low-calcium diet (trabecular bone volume loss of 19%), ovariectomy (53%) and ovariectomy combined with low-calcium diet (71%) were observed. In all the analyzed groups, the percentage loss (as compared with controls) of transversal trabeculae was more significant than the loss of longitudinal trabeculae. In the LCa group, transversal trabecular loss was 39%, longitudinal 25%, in (Ovx): 63% and 54%, respectively, and in OVX + LCa: 77% and 72%. The structural anisotropy coefficient, defined as the ratio of transversal to longitudinal trabecular surface area was 0.64 for (N), 0.50 for LCa, 0.49 for OVX, and 0.54 for OVX+LCa groups. CONCLUSIONS: The effect of ovariectomy and low-calcium diet on trabecular structure can be assessed quantitatively by means of analysis of transversal and longitudinal trabeculae associated with the main direction of strain. The degree of transversal trabecular loss is much higher than the longitudinal trabecular loss; the difference becomes smaller with the progress of bone destruction, being greatest in the LCa group, the smallest in the (OVX+ LCa) group.

Assessment of effects of lanthanum carbonate with and without phosphate supplementation on bone mineralization in uremic rats.

AIMS: Previous studies have indicated that impaired bone mineralization in 5/6 th nephrectomized rats given high doses of lanthanum carbonate is due to phosphorus depletion caused by excessive binding to, and reduced absorption of, dietary phosphate. This study aimed to test this hypothesis by: 1) directly comparing the effects of a supratherapeutic dose of lanthanum carbonate or dietary phosphorus restriction on bone mineralization in a rodent model of chronic renal failure (CRF); and 2) investigating whether phosphorus supplementation would prevent the bone mineralization defect associated with lanthanum carbonate treatment. METHODS AND MATERIALS: Male Sprague-Dawley rats were subjected to sham surgery or a two-step 5/6th nephrectomy to induce CRF and randomized across five treatment groups: sham, CRF, CRF + dietary phosphorus deficiency, CRF + lanthanum carbonate (1000 mg/kg/ day), and CRF + lanthanum carbonate + parenteral phosphorus repletion. RESULTS: Rats with 5/6th nephrectomy had elevated serum creatinine, blood urea concentration, and urine volume and protein, consistent with impaired renal function, and increased urinary phosphorus and serum parathyroid hormone, consistent with hyperparathyroidism. Lanthanum carbonate and dietary phosphate insufficiency induced parallel changes in serum and urine markers of phosphate homeostasis and increased osteoid formation. These changes induced by lanthanum carbonate were normalized by systemic phosphate supplementation. CONCLUSIONS: These findings provide further support for the concept that supratherapeutic doses of lanthanum carbonate induce effects on bone mineralization in uremic rats via an indirect pharmacological mechanism (phosphate depletion) and not via direct bone toxicity.

Comparative assessment of bone among wild-type, restricted ovulator and out-of-production hens.

1. The aim of this study was to assess bone characteristics in restricted ovulator (RO) hens. These hens generally are unable to ovulate due to a point mutation in the oocyte VLDL receptor gene whose protein product mediates the uptake of yolk precursors. Because these hens do not have the cyclic calcium (Ca) metabolism associated with egg formation, they could be a useful model for studying bone metabolism. 2. RO hens had greater humerus, femur and tibia ash concentrations than wild-type (WT) and out-of-production (OP) hens. Bone mineral content and density obtained with dual-energy X-ray absorptiometry (DXA) were highly correlated with the results of conventional bone assays. 3. Gross and histological examination of the femurs confirmed the presence of extremely dense medullary bone deposition in the RO hens. However, the composition of non-collagenous protein extracts of medullary bone was similar for the two genotypes. 4. Analysis of medullary bone extracts for glycosaminoglycans (GAG) confirmed the presence of large amounts of keratan sulphate (KS) in the matrix of medullary bone. 5. Plasma Ca, total GAG and KS concentrations of RO hens were markedly higher than WT and OP hens. The changes in plasma calcium and keratan sulphate are probably a reflection of elevated Ca-binding yolk precursor molecules and intensive medullary bone formation in response to increased plasma oestrogen observed by others in RO hens.

Relations between individual and neighborhood-based measures of socioeconomic position and bone lead concentrations among community-exposed men: the Normative Aging Study.

To examine the association between lead exposure and both individual and geographic area indicators of socioeconomic position, the authors measured tibia lead concentration, a biomarker of cumulative lead exposure, using K x-ray fluorescence in a cross-sectional survey of 538 white males aged 50-92 years who were healthy when enrolled in the Normative Aging Study (Boston, Massachusetts) in the 1960s. Data on individual risk factors, education, occupation, and income were collected by questionnaire. Using subjects' residential addresses at the time of the tibia lead measurements, the authors obtained geographic area-specific measures of education, social class, and poverty by linking records to 1990 US Census block group data. In multivariate linear regression analysis controlling for age and cumulative smoking, tibia lead concentrations were 10.39 microg/g (95% confidence interval (CI) 7.80-12.97) higher in men who did not graduate from high school than in men with > or =4 years of college. Among the former men (non-high school graduates), living in an undereducated area was associated with a 9.28 microg/g (95% CI 1.59-16.97) increase in tibia lead level compared with living in a non-undereducated area; among the latter men (college graduates), no difference existed by residential area education (beta = 0.72, 95% CI -5.35 to 6.78). The authors conclude that the influence of individual socioeconomic position on cumulative lead exposure is modified by geographic area conditions.

Bone mineral assessment by dual-energy X-ray absorptiometry in patients with coxarthrosis.

Forty patients with unilateral osteoarthritis of the hip were studied with dual energy X-ray absorptiometry to quantify disuse osteopenia in their affected leg by examining the proximal femur and tibia. Bone loss was assessed as a percentage of the contralateral value which compares bone mineral density of the affected and normal sides. The percentage contralateral value in the femoral neck and Ward's triangle was 113% and 118% respectively, while that in the tibia was 75%. Bone loss in the proximal tibia of the affected leg could be of value in assessing gait since it correlates with the gait parameters on the hip rating scale. By contrast, bone mineral increase in the proximal femur and correlates only with the degree of valgus deformity of the femoral neck.

[Medical and biological assessment of a new mineral food supplement]. / Mediko-biologicheskaia otsenka novogo pishchevogo mineralnogo obogatitelia.

Egg shell was used for mineral enrichment of ration for animals. Such ration exerts favourable influence on lipid, protein, and mineral metabolism, has hemostimulating and hypoallergic effects. Egg shell was recommended as a source of calcium for enrichment of different foodstuffs.

Assessment of exposure to parenteral and oral aluminum with and without citrate using a desferrioxamine test in rats.

The primary purpose of this study was to determine the relative usefulness of various measures to monitor body aluminum burden in weanling rats fed various amounts of aluminum (0.39 mumol Al/g diet for 29 days, approximately 40 mumol Al/g diet with or without citrate for 29 days and approximately 100 mumol Al/g diet with citrate for 12 or 29 days) or injected intraperitoneally with graded doses of aluminum (0.01, 4.6, 11.8, 23.5 or 94 mumol Al). Twenty-four hours prior to sacrifice, all rats were injected intraperitoneally with either desferrioxamine (75 mg DFO) or buffer. All seven indices of aluminum exposure monitored (i.e. tibia, liver, kidney and serum aluminum concentrations; changes in serum aluminum concentrations in response to DFO; urinary aluminum excretion with and without DFO treatment) were highly (P < 0.001) correlated to parenteral aluminum exposure. Five of these measures (i.e. tibia, liver and serum aluminum concentrations and urinary aluminum excretion with and without DFO treatment) were also highly (P < 0.001) correlated to oral aluminum loads. Changes induced by DFO were very small. Moreover, the 'DFO stimulated' serum and urine aluminum concentrations were not more correlated to the body load of aluminum, as indicated by tibia aluminum concentrations, than baseline values. Comparisons of aluminum exposure in tibias and sera of rats exposed to parenteral and oral aluminum indicated that only 0.01-0.04% of dietary aluminum was absorbed. Ingestion of citrate had small but significant effects on aluminum retention.

Autoradiographic and liquid scintillation assessment of beta-amino-propionitrile-induced inhibition of collagen maturation using 3H-proline and 3H-lysine.

BAPN (0.1 mg/day) was injected into chick embryos for 4 days starting on the 7th day of incubation. On the 11th day, the embryos were administered either 3H-proline or 3H-lysine. 36 h later, the incorporation of each isotope by the periosteal osteogenic cells as well as into bone matrix was investigated by autoradiography. The incorporation of the two isotopes into whole bones was assessed by liquid scintillation counting. 3H-proline incorporation into the cellular or matrical compartments was unaffected by treatment. As compared to the controls, 3H-lysine label in BAPN-treated embryonic bones was significantly higher in the cellular compartment but was reduced over the bone matrix. The data provide the first direct morphological evidence that BAPN probably induces certain changes in the maturation of collagen involving lysyl residues which result in an inhibition of cross-linkage formation in collagen.

Assessment of the quantity of biologically available phosphorus in yeast RNA and single-cell protein.

Two assays were conducted with chicks fed a phosphorus-deficient crystalline amino acid diet from day 8 to 17 posthatching to evaluate the phosphorus availability of purified torula yeast RNA and single-cell protein (Torula yeast). Tibia bone ash of the chicks was the criterion used in both assays, and regression analysis was used to evaluate bone ash as a function of phosphorus intake. A standard curve was established by feeding graded levels of KH2PO4 in each assay. Weight gain, tibia weight and tibia ash all responded to phosphorus supplementation whether provided by KH2PO4, RNA or single-cell protein. Weight of tibia ash provided the best linear fit when evaluated as a function of phosphorus intake. Availability in both assays estimated using the slope-ratio technique wherein milligrams of available phosphorus per gram of RNA or single-cell protein was calculated. In assay 1 the available phosphorus in RNA was found to be 93.2 mg/. In assay 2 the available phorphorus in single-cell protein was found to be 14.1 mg./g.