Two-site assays of bone gla protein (osteocalcin) demonstrate immunochemical heterogeneity of the intact molecule.

We developed a panel of monoclonal antibodies to human bone gla protein (BGP; osteocalcin) peptides that span the linear sequence of the molecule, specifically BGP 1-12 (N-terminal), BGP 15-30 (midregion), and BGP 38-49 (C-terminal). These antibodies were evaluated in various combinations of two-site formats in studies of serum BGP concentrations. For clinical studies, we selected from a panel of antibodies the two most sensitive antibody pairs for the intact molecule (N-C); we also used a polyclonal RIA based on BGP-C. For the two-site format, we used two N-terminal antibodies, 029 and 052, adsorbed to polystyrene beads, and radioiodinated a C-terminal antibody, 663. The standard for each of the assays was purified human BGP. The following BGP serum concentrations (microgram/L, mean +/- SE) were measured with the various assays: by the 029-663 assay, results for normal subjects were 7 +/- 3, for patients with renal failure 25 +/- 8, and for patients with Paget disease 12 +/-4; by the 052-663 assay, the respective results were 22 +/- 4, 44 +/- 12, and 31 +/- 7; by the polyclonal assay, the results were 3 +/- 0.2, 13 +/- 2, and 5 +/- 1. The two intact (N-C) assays were significantly (P < 0.01) correlated (r = 0.94), but their serum values differed by more than twofold in terms of the same BGP standard. The polyclonal assay significantly correlated with each of the intact assays (r = 0.83, 0.77), but it, too, gave different serum values for BGP. These studies demonstrate the immunochemical heterogeneity of circulating BGP, heterogeneity that is manifest even in immunoassays specific for the same region of the molecule.     

New biochemical marker for bone metabolism. Measurement by radioimmunoassay of bone GLA protein in the plasma of normal subjects and patients with bone disease.

gamma-Carboxyglutamic acid-containing protein of bone (BGP) is an abundant noncollagenous protein of mammalian bone. BGP has a molecular weight of 5,800 and contains three residues of the vitamin K-dependent amino acid, gamma-carboxyglutamic acid. We have applied a radioimmunoassay based on calf BGP for the measurement of the protein in the plasma of 109 normal humans and 112 patients with various bone diseases. BGP in human plasma was demonstrated to be indistinguishable from calf BGP by assay dilution studies and gel permeation chromatography. The mean (+/- SE) concentration of BGP in normal subjects was 6.78 (+/- 0.20) ng/ml, 7.89 (+/- 0.32) for males and 4.85 (+/- 0.35) for females. Plasma BGP was increased in patients with Paget's disease of bone, bone metastases, primary hyperparathyroidism, renal osteodystrophy, and osteopenia. Plasma BGP did correlate with plasma alkaline phosphatase (AP) in some instances, but there were dissociations between the two. It was additionally observed that patients with liver disease had normal plasma BGP despite increased plasma AP, a reflection of the lack of specificity of AP measurements for bone disease. Our studies indicate that the radioimmunoassay of plasma BGP can be a useful and specific procedure for evaluating the patient with bone disease.     

Determination of bone Gla protein (osteocalcin) by enzyme-linked immunosorbent assay

Bone Gla protein (BGP, osteocalcin) is a marker of bone formation. We present a novel enzyme-linked immunosorbent assay for measuring BGP in plasma and serum. The antibody used was raised in rabbits following immunisation with highly purified bovine BGP conjugated with keyhole limpet hemocyanin. The binding of the antibody to BGP was calcium-dependent. The sensitivity, inaccuracy, and imprecision of the assay equal or exceed existing radioimmunoassays, and the present assay is less tedious. Plasma BGP in 249 healthy adults, aged from 20 to 93 yr, was 8.3 +/- 6.6 micrograms/l (mean +/- 2 SD). A significant decrease was seen in both sexes from the third to the forth decade of life. A subsequent significant increase was seen with age in women. but not in men. Plasma BGP was significantly higher in young men than in young women, and significantly higher in elderly women than in elderly men. Values were in the same range as those found with existing radioimmunoassays.     

Binding of Antibiotics to Bovine and Ovine Serum

The degree of binding of 37 antibiotics to bovine and ovine serum, after treatment at therapeutic doses, was determined by equilibrium dialysis and ultrafiltration methods. In general, binding values obtained by the two methods were comparable. The extent of binding varied from 0% for cephaloridine and kanamycin to >95% for novobiocin and fusidic acid. Of the 37 drugs studied, one-fourth were less than 25% bound, one-fourth were more than 75% bound, and the percentage binding of about half of the antibiotics ranged from 25 to 75%. Animal to animal variations in the extent of binding of a particular antibiotic were very small. The capacity of bovine or ovine serum to bind antibiotics was, with a few exceptions, similar to the reported capacity of human serum. At drug concentration ranges usually achieved during therapy, variations in drug levels in serum did not influence the degree of binding except with cephalexin, lincomycin, clindamycin, and chloramphenicol. With these antibiotics, the extent of binding increased two- to sevenfold with the decrease in drug concentration in serum.     

Characterization and assay of tartrate-resistant acid phosphatase activity in serum: potential use to assess bone resorption

We improved the spectrophotometric assay of tartrate-resistant acid phosphatase (TrACP; EC 3.1.3.2) activity in serum. During development of the assay we found that human serum contains a dialyzable, mixed-type noncompetitive inhibitor(s) of TrACP activity, the effects of which on the assay were substantially lessened by diluting the serum sample with water before assay and increasing the substrate concentration. Hemolysis releases into serum a significant amount of TrACP activity from erythrocytes, which can be inactivated by incubating the serum at 37 degrees C for 1 h before assay. Our improved assay was reproducible (CV = 5%), and measured within 10% of the amount of added bovine skeletal TrACP activity. Preliminary application of the assay revealed that the amount of serum TrACP activity in patients with skeletal diseases differed from normal values and changed in the same direction as the expected change in bone turnover, suggesting that TrACP activity in serum could be useful clinically as a marker of bone metabolism, possibly of bone resorption.     

骨质疏松、非重力骨折患者血清BGP、PINP含量及其临床意义的探讨

本文观察了正常人、骨质疏松和非重力骨折患者的血清BGP和PINP含量。正常人组、骨质疏松组和非重力骨折组的血清BGP含量(±s,ug/L)分别为8.98±3.24(n=40),18.92±12.10(n=51)和7.49±4.86(n=30),血清PINP的含量分别为42.395±21.35(n=37),52.725±21.17(n=56)和52.37+29.27(n=37)。骨质疏松组的血清BGP显著高于正常人组和非重力骨折组,有非常显著的意义(P<0.01),非重力骨折组的血清BGP低于正常人组,无显著性意义(P>0.05)。骨质疏松组的血清PINP高于正常人组有显著性意义((P<0.05),非重力骨折组的血清PINP明显高于正常人组组无显著性意义(P>0.05)。频率分析显示,三个组的血清BGP的分布存在很大差异,骨质疏松组在高值区显著增多,占43.1％;非重力骨折组低值增多,占30％。三个组的血清PINP的频率分布重迭面很大,高达80％。相关分析表明,骨质疏松组和非重力骨折组的血清BGP和PINP的相关性都非常显著(P<0.01)。     

Assessment of bone turnover in postmenopausal osteoporosis by measurement of serum bone Gla-protein

Controversy persists regarding the abnormality of bone turnover responsible for bone loss in women with postmenopausal osteoporosis. To evaluate this, we measured serum bone Gla-protein (BGP), a specific marker for bone turnover, in 62 untreated patients with postmenopausal osteoporosis. Results were compared with those in 142 normal women and were expressed as standard deviations from the age-adjusted predicted mean (Z score). Serum BGP was increased (+0.48 S.D., p = 0.002) in the osteoporotic patients; 9.7% of patients were greater than 2 S.D. above but none were greater than 2 S.D. below the normal mean. Moreover, when data from normal postmenopausal women (ages 51 to 75 years) and the osteoporotic patients were merged, significant negative correlation existed (r = -0.36, p less than 0.001) between serum BGP and bone density of the lumbar spine assessed by dual photon absorptiometry. Serum alkaline phosphatase, a less specific marker for bone formation, was also increased (+0.96 S.D., p less than 0.001) in the osteoporotic patients. The data suggest that overall bone turnover is increased in patients with postmenopausal osteoporosis and do not support the concept that an absolute decrease in bone formation is the major cause of the bone loss.     

Increase in serum bone gamma-carboxyglutamic acid protein with aging in women. Implications for the mechanism of age-related bone loss.

Because it is unclear whether age-related bone loss results from increased bone resorption, decreased bone formation or both, we measured the serum level of bone Gla-protein (BGP), a specific marker for bone turnover, in 174 women, ages 30 to 94 yr. Serum BGP increased linearly with aging (r = 0.44, P less than 0.001) from 4.4 +/- 0.4 (mean +/- SE) in the 4th decade to 8.9 +/- 0.9 ng/ml in the 10th decade. This increase correlated inversely (P less than 0.001) with concomitant decreases in bone mineral density at the lumbar spine, midradius, and distal radius. Using partial correlation coefficients, serum BGP still correlated positively with age (r = 0.31, P less than 0.001) after creatinine clearance was fixed but not with creatinine clearance (r = -0.04, NS) when age was fixed. Urinary hydroxyproline (r = 0.29, P less than 0.001), an index of bone resorption, and serum alkaline phosphatase (r = 0.31, P less than 0.001), an index of bone formation, also increased with age and these increases correlated with increases in serum BGP (r = 0.39, P less than 0.001 and r = 0.43, P less than 0.001, respectively). Serum immunoreactive parathyroid hormone concentrations (r = 0.39, P less than 0.001) and urinary cyclic AMP excretion (r = 0.38, P less than 0.001) increased, suggesting that PTH secretion increased with age; these increases correlated significantly with increases in serum BGP. A subgroup of 32 women who were found to have vertebral fractures, hip fractures, or both had significantly higher values for serum BGP than the remainder. These data suggest that overall bone turnover increases in women with aging and, especially considering the concomitant decrease in skeletal mass, do not support the view that age-related bone loss results primarily from decreased bone formation.     

人工虎骨对原发性骨质疏松症患者骨钙素和抗酒石酸酸性磷酸酶5b的影响

目的观察人工虎骨对原发性骨质疏松症患者骨代谢生化标志物骨钙素(BGP)和抗酒石酸酸性磷酸酶5 b(TRACP 5b)水平的影响。方法入选原发性骨质疏松症患者86例,性别均为女性,绝经1年以上,年龄(60.5±3.6)岁。口服人工虎骨粉胶囊,3次/d,每次3粒,共3个月。治疗前后分别测定患者血清BGP水平(放射免疫法)和TRACP5b水平(酶联免疫吸附法)。结果治疗后血清BGP明显增高[(8.31±2.41)]μg/L vs(5.81±1.93)μg/L,P<0.05],血清TRACP 5b明显降低[(2.49±0.81)]u/L vs(4.26±0.86)u/L,P<0.05]。结论人工虎骨可以通过增加原发性骨质疏松症患者的骨形成,减少骨吸收而发挥其治疗作用。     

Radioimmunoassay for 1,25-dihydroxyvitamin D in serum or plasma

For this sensitive RIA for 1 alpha,25-dihydroxyvitamin D, we used antibodies to 1 alpha,25-dihydroxycholecalciferol-3-hemisuccinate conjugated to bovine serum albumin, raised in rabbits. These antibodies show a high affinity for 1 alpha,25-dihydroxyvitamin D3 but cross react with other vitamin D metabolites as well. Extraction and liquid chromatography are required to isolate the 1 alpha,25-dihydroxyvitamin D from human serum or plasma with benzene (analytical recovery, 96%) and chromatographed by "high-performance" liquid chromatography on a silica column. The sensitivity of the assay is 2 pg/tube. The between-assay CV is 12.6% (means = 61.8 ng/L, n = 27) and the within-assay CV is 7.4% (means = 61.8 ng/L, n = 15). The reference interval, obtained from data on serum of healthy persons, is 51.8 +/- 15.1 ng/L (means +/- SD, n = 91), with no sex-related difference. Patients with chronic renal failure have lower concentrations in serum, and patients suffering from calcium nephrolithiasis have increased concentrations. Our assay offers the advantage of a relatively short prepurification procedure and the production of large amounts of stable gamma-globulins that can be used for several years without loss of binding properties.     

Clinical assessment of collagen cross-linked N-telopeptides as a marker of bone metabolism in patients with primary hyperparathyroidism.

This study assesses the clinical usefulness of collagen cross-linked N-telopeptides (NTx), a new marker for bone resorption, as a marker for primary hyperparathyroidism. The study was conducted on 114 patients who had undergone parathyroidectomy for primary hyperparathyroidism. Urine NTx levels were longitudinally determined by ELISA. Longitudinal serum bone Gla protein (BGP) determinations and dual energy X-ray absorptiometry (DEXA) examinations were also performed before and after parathyroidectomy. Before parathyroidectomy, the mean urine NTx level in primary hyperparathyroidism was 149.0 +/- 116.8 pmol BCE/mumol Cr (normal: < 55 pmol BCE/mumol Cr), and the positive rate was 88.5%. The mean BGP level was 33.5 +/- 59.1 ng/mL (normal: 12.7 ng/mL), and the positive rate was 77.7%. The NTx positive rate was higher than the BGP positive rate. NTx values were significantly correlated with bone mineral density (P < 0.002) and BGP values (P < 0.005). Two weeks after parathyroidectomy the NTx levels had already decreased significantly to 65.1 pmol BCE/mumol Cr (P < 0.001), and by one month postoperatively they had fallen to 55.9 pmol BCE/mumol Cr, reaching the normal range much sooner than BGP. The NTx normalization rate two weeks postoperatively was 37%, and by 12 months postoperatively it was normal in 83% of patients. BGP levels decreased more gradually than the NTx levels, and only became normal in 9% of cases two weeks postoperatively, as opposed to 74% by 12 months. It is concluded that NTx reflects bone turnover in primary hyperthyroidism, and that it is an excellent marker for bony lesions before and after parathyroidectomy.     

中医健脾单元疗法对强直性脊柱炎患者的疗效及对血清骨钙素、抗酒石酸酸性磷酸酶的影响

目的探讨中医健脾单元疗法对强直性脊柱炎(AS)患者疗效及对血清骨钙素(BGP)、抗酒石酸酸性磷酸酶(TRACP)的影响。方法按随机数字表将60例强直性脊柱炎患者分成治疗组(34例)和对照组(26例),治疗组采用中医健脾单元疗法,即新风胶囊+中医辨证论治+中药熏蒸治疗,对照组采用柳氮磺嘧啶(SASP)+中医辨证论治+中药熏蒸治疗。结果①中医证候疗效评价结果示两组总有效率(65.4%比88.2%)差异有统计学意义(P<0.05)。②两组治疗后AS患者血清BGP水平升高,同时TRACP降低(P<0.01),而治疗组治疗后血清BGP水平显著高于对照组,TRACP显著低于对照组(P<0.01)。结论中医健脾单元疗法能够提高AS患者的生活质量,改善临床症状体征及骨代谢水平。     

Artefactual increase in serum thyrotropin concentration caused by heterophilic antibodies with specificity for IgG of the family Bovidea

A 19-year-old white woman presented with symptoms compatible with mild hyperthyroidism and biochemical evidence suggestive of autonomous thyrotropin (TSH) secretion. Intensive investigation of the pituitary-thyroid axis suggested that the basal concentrations of TSH were artefactually increased owing to heterophilic antibodies in the patient's serum with a broad specificity for immunoglobulin class G of the family Bovidea. These heterophilic antibodies complexed with the ovine antisera to human thyrotropin that are used in the RIA system, in particular blocking the binding of TSH but also partly blocking interaction with the second antibody. When TSH was measured immunometrically or by an RIA with TSH-specific antisera of rabbit origin, the concentrations measured were within the appropriate reference intervals. The blocking effect can be overcome by including large quantities of non-TSH-specific ovine IgG in assay incubation mixtures. Interference of this type is generally not appreciated and its incidence is poorly characterized, but it may have implications for any method in which antibodies are used as reagents.     

慢性肾脏病3~5期患者骨代谢相关指标分析

目的探讨慢性肾脏病(CKD)3~5期患者血清骨代谢相关指标水平的变化情况,为继发性骨代谢疾病的早期诊断与预防提供依据。方法回顾性分析本院收治的确诊为CKD 3~5期的261例患者血清中甲状旁腺激素(iPTH)、钙(Ca)、磷(P)、骨钙素(BGP)、降钙素(CT)、骨特异性碱性磷酸酶(OST)、25-羟基维生素D[25(OH)D]等指标水平的变化,并与反映肾脏功能的血清尿素(Urea)、尿酸(UA)、血肌酐(SCr)等指标进行相关性分析。结果在CKD 3~5期,血清iPTH和P水平进行性升高,差异有统计学意义(P0.05);BGP和CT在CKD5期明显升高,差异有统计学意义(P0.01)。Spearman相关分析显示,iPTH、P与Urea、UA、SCr均呈正相关(P0.05)。结论 CKD 3~5期iPTH、P、BGP等指标有差异,并与Urea、UA和SCr等指标呈正相关,可辅助诊断骨代谢疾病。     

Plasma BGP: an indicator of spontaneous bone loss and of the effect of oestrogen treatment in postmenopausal women

One hundred and ninety-one healthy early postmenopausal women, aged 45-54 years, were randomized to 2 years of treatment with (a) percutaneous 17 beta-oestradiol combined with progesterone (n = 29) or placebo (n = 28); (b) oral oestradiol valerate combined with cyproterone acetate (n = 37) or placebo (n = 39); (c) 24R, 25 (OH)2D3 (n = 29) or placebo (n = 29). We measured the plasma bone Gla-protein (BGP), bone mineral content of the proximal forearms (BMC), bone mineral density in the spine (BMDspine) and total body bone mineral (TBBM) in all the women before, and during, the study. In the groups of women receiving the oestrogen preparations, the plasma BGP decreased highly significantly (P less than 0.001) to a premenopausal level. The initial plasma BGP concentration was significantly related to the loss of BMC (P less than 0.001) in the placebo groups. The changes in plasma BGP were an indicator of the oestrogen response on BMC. We conclude that serial determinations of plasma BGP are useful for determination of the effect of oestrogen therapy in groups of patients, and that plasma BGP measured at the time of the menopause indicates what the rate of bone loss will be.     

Bone alkaline phosphatase measured with a new immunoradiometric assay in patients with metabolic bone diseases

We have investigated the clinical utility of a new quantitative two-site radioimmunometric assay specific for bone alkaline phosphatase (B-ALP) in 219 healthy control subjects and in 264 patients with various metabolic bone diseases. B-ALP was compared with total alkaline phosphatase (T-ALP) and with osteocalcin (BGP). B-ALP increased linearly with age in both sexes. In postmenopausal normal women B-ALP increased by 82% compared with premenopausal normal women, whereas the differences between pre- and postmenopausal women for T-ALP and BGP were 18% and 30% respectively. As assessed by Z -score, the highest values of B-ALP were found in patients with Paget's disease of bone, bone metastases or hyperparathyroidism and in patients on maintenance haemodialysis. In osteoporotic patients, B-ALP, but not T-ALP, showed a slight but significant ( P  < 0.05) difference compared with normal women. On the basis of bone turnover, osteoporotic patients were divided into two groups: high turnover and low turnover; B-ALP, like BGP, was significantly ( P  < 0.01) higher in patients with high turnover. In conclusion, B-ALP, measured by this new method, can be considered a sensitive marker of bone turnover and could be especially useful in identifying women at risk of developing osteoporosis.     

Antibodies to dietary antigens in rheumatoid arthritis--possible molecular mimicry mechanism.

Antibodies in serum from some patients with rheumatoid arthritis, recognize bovine albumin present in the milk, as determined by immunoprecipitation analysis from 125I-milk extracts. This antigen was also immunoprecipitated from bovine sera. These and ELISA studies showed that BSA is preferentially recognized over other proteins present in the milk. Panel studies demonstrated that although the average reactivity for BSA was high, only one third of the sera tested displayed a reactivity above the mean. The possibility of a molecular mimicry mechanism in RA between this food antigen and other human antigens was investigated. A sequence alignment analysis showed that the residues 141-157 of bovine albumin significantly differed from the corresponding fragment of human albumin, but were highly homologous with human collagen type I, C1q and vitamin D binding protein. In support of the immunogenicity of this fragment, we found that representative RA sera displayed a specific reactivity for a synthetic peptide containing the BSA residues responsible for the homology. Furthermore, most of the epitopes recognized on BSA by the RA sera seem to be conformationally dependent as heat denaturation or reduction followed by alkylation lead to a diminished recognition.     

The effects of fimbriate and non-fimbriate strains of Escherichia coli on the oxidative reactions of neutrophils from a variety of species

5 fimbriate strains and 5 non-fimbriate strains of Escherichia coli were tested for their ability to enhance the cyanide insensitive oxygen consumption of human, bovine and ovine blood neutrophils in the absence of serum opsonins. Neutrophils from all 3 animal species showed significantly increased oxygen consumption when challenged with fimbriate strains. Only ovine neutrophils were stimulated by non-fimbriate strains although to a lesser extent than by fimbriate strains. In the presence of serum the stimulation of ovine neutrophils was the same whether fimbriate or non-fimbriate strains were used.     

Plasma bone Gla protein concentrations in healthy adults. Dependence on sex, age, and glomerular filtration

Plasma bone Gla protein (BGP) was determined by radio-immunoassay in 266 healthy adults, men (n = 132) and women (n = 134), aged 20-79 years. In the women aged 30-69 years, plasma BGP increased significantly with age (r = 0.44, p less than 0.001), and a particularly steep increase was seen from 1.1 +/- 0.5 (mean +/- 1 SD) in the fifth decade to 2.0 +/- 1.4 nmol/l in the seventh decade. In men, aged 30-69 years, no correlation was found between plasma BGP and age (r = -0.07, NS). Plasma bone Gla protein is removed from the circulation mainly by the kidneys and the increased plasma BGP in the women could be caused by decreased renal clearance. The interrelationship was analysed by means of partial correlation. When creatinine clearance was held constant in women, BGP still correlated positively with age (r = 0.40, p less than 0.001), but not with creatinine clearance (r = 0.003, NS) when age was fixed. Plasma BGP was significantly increased above normal in 35 patients with chronic renal failure (10.2 +/- 14.6 nmol/l). Non-linear regression analysis showed that plasma BGP was within the normal range when 24-h creatinine clearance was greater than 30 ml/min, and large increases in plasma BGP did not occur until the 24-h creatinine clearance was below 20 ml/min. We conclude that, in normal subjects and patients with mild to moderate renal failure, plasma elevations of BGP reflect increased bone turnover rather than decreased renal clearance.     

New immunoenzymatic assay for human thyrotropin compared with two radioimmunoassays

We have evaluated a new immunoenzymatic assay for human thyrotropin involving three monoclonal antibodies (Abbott HTSH EIA) and compared the results with those of two conventional nonequilibrium double-antibody radioimmunoassay (RIA) methods: Clinical Assays' RIA and a research RIA (J Clin Endocrinol Metab 1975; 41:676). Mean values for thyrotropin in 100 euthyroid serum samples were similar in the Abbott and Clinical Assays methods, but both sets were significantly higher than those by the research RIA. By all methods, values for hypothyroid patients were clearly higher than values for euthyroid subjects. Results for hyperthyroid and euthyroid subjects were resolved slightly better with the research RIA than with the Abbott kit. The new Abbott assay was far more sensitive than either the Clinical Assays RIA or our research RIA. The correlation of results of the Abbott assay with those of the Clinical Assays and the research RIA exceeded 90% for samples from hypothyroid patients. The Abbott assay replaces radioisotope counting with spectrophotometric detection.