Cultures of human osteoblastic cells from dialysis patients: influence of bone turnover rate on in vitro selection of interleukin-6 and osteoblastic cell makers.

The factors contributing to renal osteodystrophy are still incompletely characterized. A variety of cytokines and growth factors appear to have ill-defined roles in this disease. Our aim is to compare osteoblastic cell growth and different osteoblastic markers in vitro with histomorphometric bone parameters and some serum bone-turnover markers in vivo in dialysis patients with either high- (HTBD) or low-turnover (LTBD) bone disease. Six patients were diagnosed to have LTBD, and another five patients, HTBD. Intact parathyroid hormone (PTH) and osteocalcin (OC) levels in serum were greater in patients with HTBD than in those with LTBD. Osteoblastic cells isolated from iliac crest biopsy specimens were grown in culture medium for different times up to 13 days. Osteoblastic cell growth (cell number and area under the cell growth curve) was greater in patients with HTBD than in those with LTBD. Static and dynamic bone formation parameters correlated with serum PTH levels. No correlation was found between PTH and osteoblastic cell proliferation. OC, C-terminal type I procollagen, and alkaline phosphatase osteoblastic secretion in vitro were similar in the HTBD and LTBD groups. However, interleukin-6 (IL-6) secretion was greater in cells isolated from patients with LTBD. Our results indicate that osteoblastic cell growth and osteoblastic IL-6 secretion are related to bone turnover in patients with osteodystrophy. Our findings support the hypothesis that factors other than PTH level might have an important role in affecting osteoblastic function in renal osteodystrophy.

[Methods to assess the ex vivo growth capacity of mesothelial cells obtained directly from peritoneal effluent]. / Metodología para evaluar la capacidad de crecimiento ex vivo de las células mesoteliales obtenidas directamente del efluente peritoneal.

The anatomical and functional integrity of mesothelial cells (MC) is necessary for peritoneal membrane stability. At present, there is no satisfactory method to assess MC function and regenerative capacity in individual peritoneal dialysis (PD) patients. MC may be cultured from peritoneal biopsy specimens, but peritoneal biopsy is an invasive procedure that cannot be performed serially. The aim of this study is to explore the feasibility of serial culture of MC from the peritoneal effluent of PD patients. Fifty-two randomly selected PD patients were studied. MC were obtained from the peritoneal effluent of nocturnal 2.27% glucose exchanges and cultured in T25 tissue culture flasks. Subconfluent MC cultures were obtained in 80.7% of patients. At this stage, the percentage of cells in the tissue cultured flask characterized as MC by morphology and immunostaining had increased to 95.5%. MC were then subcultured in multi-well culture plates, where they showed exponential cell growth until day 16. Nine (17%) patients released low numbers of MC into the effluent and MC could not be cultured to subconfluence. One additional patient released and apparently adequate number of MC that repeatedly failed to reach confluence. Patients showed the same behavior in several cultures performed. In conclusion, peritoneal MC released into peritoneal effluent are accessible for profound analysis by a culture technique. This technique opens the possibility of serial follow-up of the biology of MC individual PD patients.

Influence of skeletal site of origin and donor age on osteoblastic cell growth and differentiation.

Bone loss with aging may be due, at least in part, to inadequate bone formation. Moreover, the process of bone aging is known to follow a different pattern throughout the skeleton. In this study, we examined the cell proliferation rate (area under the cell growth curve, AUC) and the secretion of C-terminal type I procollagen (PICP), alkaline phosphatase (ALP), and osteocalcin (OC) in primary cultures of osteoblastic cells from human trabecular bone. Osteoblastic cells were obtained for 168 donors (100 women and 68 men). Ninety-eight bone samples were obtained from subjects undergoing knee arthroplastia, 52 aged 50-70 years (64 +/- 5) and 46 over age 70 (73 +/- 2). Another 70 bone samples were obtained from subjects undergoing hip arthroplastia; 51 were 50-70 years old (64 +/- 4) and 19 were over 70 (75 +/- 5). Osteoblastic cells from the older donors had a lower proliferation rate and OC secretion than those from younger subjects. However, ALP secretion was higher in the former subjects, whereas PICP secretion was unchanged. Osteoblastic cells from hip had a lower proliferation rate than those from knee. PICP secretion was also lower and ALP secretion was higher in the former cells. In age-matched cell cultures, osteoblastic cells from the knee had higher proliferation rate and PICP secretion than osteoblastic cells from the hip. However, ALP secretion was lower in knee osteoblastic cells than those from hip only in the younger group. With aging, ALP secretion was found to increase in knee osteoblactic cells, whereas OC secretion decreased in osteoblastic cell cultures from the hip. Our findings suggest that bone loss with aging may be accounted for, at least in part, by a decreased osteoblastic cell proliferation and an increased osteoblastic maturation. In addition, our data indicate that these changes with aging do not occur similarly at different skeletal sites.

Influence of skeletal site of origin and donor age on 1,25(OH)2D3-induced response of various osteoblastic markers in human osteoblastic cells.

Age-related bone loss may be a consequence of a lack of osteoblastic formation and/or function. In vitro, the osteoblastic response to 1,25(OH)2D3, an important regulator of osteoblastic function, appears to depend on the stage of osteoblastic maturation. In this study, we examined the response to 1,25(OH)2D3 of C-terminal type I procollagen (PICP), alkaline phosphatase (ALP), and osteocalcin (OC) secretion in primary cultures of osteoblastic cells from human trabecular bone (hOB). Forty-four bone samples were obtained from subjects undergoing knee arthroplastia, 20 aged 50-70 (64 +/- 5), and 24 >70 (73 +/- 2) years. Another 33 bone samples were obtained from subjects undergoing hip arthroplastia, 21 were aged 50-70 (64 +/- 4) and 12 >70 (75 +/- 5) years. Pooling knee and hip hOB cell cultures, we found that PICP secretion decreased after 1,25(OH)2D3 in hOB cells from the older group (>70 years). Treatment with 1,25(OH)2D3 increased ALP secretion in these cells only in the younger group (50-70 years), whereas it increased OC secretion in hOB cells in both age groups. By pooling hOB cell cultures from both age groups we found that knee hOB cells increased OC secretion, and decreased PICP secretion, after 1,25(OH)2D3. This metabolite also increased OC secretion in hip hOB cells. Considering the influence of donor age at the same skeletal site, 1,25(OH)2D3 was found to stimulate ALP secretion only in knee hOB cells in the younger group. In contrast, this metabolite decreased ALP secretion in hip hOB cells in the older group. PICP secretion decreased after 1,25(OH)2D3 only in hOB cells in the older group, at both skeletal sites. In age-matched cultures, OC secretion was lower in hip hOB cells compared with those from the knee in the older group, but was similar in these cell cultures from both skeletal sites in the younger group. OC secretion after 1,25(OH)2D3 stimulation did not show age differences in knee hOB cells, but was lower in hip hOB in the older group. In summary, our results demonstrate that the response of various osteoblastic markers to 1,25(OH)2D3 in primary cultures of hOB cells depends on the donor age and skeletal site of origin.

[Bone metabolism in non-cholestatic chronic hepatopathy]. / Metabolismo óseo en la hepatopatía crónica no colestásica.

BACKGROUND: Osteoporosis may be associated with parenchymal hepatopathy and chronic alcoholism. Biochemical studies which are linked with bone metabolism and the bone densitometry may help to understand its physiopathology, before the symptoms appear and its consequences become inevitable. PATIENTS AND METHODS: The study of bone metabolism and densitometry has been carried out in a population of 86 males, distributed in 4 groups: group I, control (17 men), group II, patients with chronic hepatopathy without alcoholism (25 patients), group III, chronic alcoholic without hepatopathy (21 patients), and group IV, patients with chronic alcoholic hepatopathy (23 patients). The results of densitometry and biochemical parameters in relation with bone metabolism are cross checked among these 4 groups. RESULTS: We found out that patients with chronic alcoholic hepatopathy have bone mineral density (BMD), at femoral level, significatively lower than that of the other 3 groups (p < 0.05). In chronic hepatopathy, regardless of its etiology, significant alterations in biochemical parameters of bone metabolism found, consisting basically in shrinked plasmatic level of 25-hydroxivitamin-D (25-OH-D) (p < 0.05). The plasmatic levels of calcitriol, magnesium and intact parathyroid hormone (PTHi) were significantly lower in chronic alcoholic hepatopathy than in the others 3 groups (p < 0.001, p < 0.001 and p < 0.05, respectively). CONCLUSIONS: Chronic hepatopathy is associated with deficiency in vitamin D. Alcoholism added to chronic hepatopathy has a negative influence on the plasmatic levels of calcitriol, magnesium and PTHi as well as in the femur BMD. Alcoholism not associated with chronic hepatopathy is not sufficient to cause significant alterations in the studied parameters.

Effect of polyethylene on osteocalcin, alkaline phosphatase and procollagen secretion by human osteoblastic cells.

We have studied the direct effects of polyethylene particles on osteoblastic function in primary human bone cell cultures. The cells were obtained from trabecular bone fragments of patients undergoing knee reconstructive surgery. When the cells reached confluency, they were subcultured into two flasks, one untreated (control culture) and the other treated with polyethylene particles, and incubated until confluency. Osteoblastic function was evaluated by assaying osteocalcin, alkaline phosphatase, and C-terminal procollagen type I, with and without 1,25(OH)2D stimulation, in the cell-conditioned medium. We found that addition of polyethylene to these osteoblastic cell cultures induced higher levels of secreted osteocalcin after 1, 25(OH)2D stimulation. Alkaline phosphatase levels increased whereas C-terminal procollagen type I levels decreased in the cell conditioned medium after polyethylene was added to the cultures. Treatment of the control cultures with 1,25(OH)2D stimulated alkaline phosphatase levels and decreased C-terminal procollagen type I. However, these osteoblastic markers in 1,25(OH)2D-treated cells did not change in cultures with polyethylene. This study demonstrates that polyethylene particles have a direct effect on osteoblastic markers in human bone cells in culture.

C-terminal parathyroid hormone-related protein inhibits proliferation and differentiation of human osteoblast-like cells.

Parathyroid hormone-related protein (PTHrP) is synthesized by osteoblasts, although its local role in bone is not completely understood. The C-terminal (107-111) region of PTHrP seems to be a potent inhibitor of osteoblastic bone resorption. We studied the effect of this PTHrP domain on the proliferation and synthesis of osteoblastic markers in osteoblast-like cells from adult human bone. We found that the human (h)PTHrP(107-139) fragment, between 10 fM and 10 nM, inhibited 3H-thymidine incorporation into these cells. The antiproliferative effect of the latter fragment, or that of hPTHrP(107-111), was similar to that induced by [Tyr34] hPTHrP(1-34) amide, bovine PTH(1-34), and hPTHrP(1-141), while hPTHrP(38-64) amide was ineffective. Human PTHrP(7-34) amide, at 10 nM, and 1 microM phorbol-12-myristate-13-acetate also significantly decreased DNA synthesis in human osteoblast-like cells. Neither hPTHrP(7-34) amide nor hPTHrP(107-139), at 10 nM, stimulated protein kinase A (PKA) activity in these cells. Moreover, 100 nM H-89, a PKA inhibitor, did not eliminate the inhibitory effect of hPTHrP(107-139) on these cells' growth. However 100 nM calphostin C, a PKC inhibitor, blunted this effect of PTHrP(107-139). In addition to their antimitogenic effect, hPTHrP(107-139) and hPTHrP(107-111) inhibited basal and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-stimulated alkaline phosphatase activity in these cells. Both fragments, like 1,25(OH)2D3, decreased C-terminal type I procollagen secretion into the cell-conditioned medium, but osteocalcin secretion by these cells was unaffected by the C-terminal PTHrP fragments. These findings suggest that PTHrP may act as a local regulator of bone formation.

Enzymic determinations with rotating bioreactors and continuous-flow-stopped-flow processing. Determination of choline esters in pharmaceuticals.

The neuromuscular blocking agent, succinylcholine, and the neurotransmitter, acetylcholine, have been determined in pharmaceutical preparations utilizing a sensing unit comprised of a rotating bioreactor and an amperometric detector. The bioreactor consisted of a rotating disk made of Teflon (1 cm id) with cholinesterase (EC 3.1.1.8) and choline oxidase (EC 1.1.3.17) co-immobilized on the top surface of the disk. Amperometric detection of H2O2 and initial rate measurements were employed for quantification.

Remission of hypercalciuria in patients with tuberculosis after treatment.

The hypercalciuria evolution and other bone metabolism parameters were evaluated in patients with tuberculosis after treatment. Twenty-two patients with tuberculosis and 54 normal subjects were studied; they consumed an average diet (calcium intake 1000 mg/day). Ten of these patients and nine normal subjects were also studied after a low calcium diet (400 mg/calcium/day) and after a load of oral calcium of 1000 mg (calcium absorption test). The study with an average diet was performed after 1 week (basal) and 3, 6, and 12 months after the antituberculosis treatment was started; the calcium absorption test was carried out 2 weeks, 3 and 12 months after the treatment was started. On an average diet, patients with tuberculosis presented, at baseline state, lower calcidiol levels than normal controls. Serum calcitriol levels at baseline were higher than at 6 and 12 months. Serum parathyroid hormone (PTH) levels in patients with tuberculosis were lower than in normal controls at baseline, but these levels were similar to controls at 3, 6, and 12 months after treatment. During the calcium absorption test and under basal conditions, patients with tuberculosis showed lower serum PTH and calcidiol levels in all the dietetic situations than in normal controls. However, serum calcitriol levels were higher than in controls after the restrictive diet. After 3 months of treatment, urinary calcium excretion was normal in patients with tuberculosis during the average and low diets, but higher than in control group after calcium load. After 12 months of treatment, all the biochemical parameters of the patients with tuberculosis were similar to the control group under all the dietetic situations. These data indicate that antituberculous treatment, although it may contribute to the production of some alteration in the calcium and vitamin D metabolism, basically favors the correction of disturbances associated with tuberculosis.

Alcohol biosensor based on alcohol dehydrogenase and Meldola Blue immobilized into a carbon paste electrode.

A yeast alcohol dehydrogenase amperometric carbon paste-based biosensor, with Meldola Blue as a mediator and a dialysis membrane with a very small molecular weight cut-off for protection, is described. The influence of membrane pore size on the stability and overall kinetics of the biosensor is shown using cyclic voltammetry and stationary potential measurements. The operating potential is + 50 mV vs. Ag/AgCl, KCl sat. reference electrode. Application of this device to the determination of ethanol in alcoholic beverages was achieved successfully. In these kinds of samples and at this working potential no interferences were found.

[Vitamin D levels in patients with hip fracture in Madrid]. / Concentraciones de vitamina D en pacientes con fractura de cadera en Madrid.

BACKGROUND: Vitamin D appears to be implicated in the loss of bone mass and its most severe complication is the hip fracture. A change is produced in the metabolism of vitamin D in elderly people although its study has received little attention in many countries. METHODS: With the aim of evaluating vitamin D and its metabolites in the elderly people with hip fracture in our geographic environment (Madrid, Spain), 58 patients with hip fracture over the age of 70, and 39 subjects without a fracture or the similar age were studied. Both groups were evaluated during the season of minimum solar irradiation. The plasma concentrations of intact PTH and the serum concentrations of calcidiol and calcitriol were studied in all the patients. RESULTS: The patients with hip fracture presented significantly lower concentrations of calcidiol (11.7 +/- 6.4 vs. 18.4 +/- 12.7 nmol/l) and calcitriol (60.1 +/- 24.7 vs. 76.1 +/- 25.0 pmol/l) than in elderly persons without fracture, with similar PTH values being observed in both groups (4.8 +/- 1.9 vs. 5.1 +/- 2.3 pmol/l). CONCLUSIONS: Although Madrid, Spain is considered to be a geographical area of high solar irradiation, the elderly population studied presented a vitamin D deficiency which was found to be greater in those with hip fracture.

Determination of Michaelis-Menten and inhibitor constants by an open-closed flow injection approach (Application to the alkaline phosphatase/theophylline system).

An automatic method for the estimation of Michaelis-Menten and inhibitor constants in biochemical systems was developed using an unsegmented open-closed flow manifold, and the Lineweaver-Burk and Dixon plot methods. The methods were applied to both the determination of K(m) of the enzymatic reaction catalyzed by bovine alkaline phosphatase using 4-methylumbelliferone phosphate as substrate, and the determination of the K(i) constant from theophylline as an inhibitor of this biochemical system. The data treatment and comparative results are presented. The values obtained agree with those provided by a conventional method with the biocatalyst in solution.

Effect of oral calcidiol treatment on its serum levels and peritoneal losses.

OBJECTIVES: To evaluate calcidiol serum levels in a group of continuous ambulatory peritoneal dialysis (CAPD) patients and the effect of oral calcidiol treatment on serum levels and peritoneal losses. STUDY DESIGN: Twenty patients (13 female, 7 male) were studied for 12-60 months. Their ages ranged 22-72 years (mean 46 +/- 15). Serum calcidiol, total protein and urea were determined at baseline and after the administration per os of 0.133 mcg/day of calcidiol for 10 days. At the same time, calcidiol and total protein were measured in peritoneal effluent at baseline and at 5, 10, and 40 days after starting this treatment. RESULTS: A significant and direct correlation between the calcidiol dialysis/plasma ratio and the peritoneal protein losses was found, both before and 40 days after calcidiol administration when calcidiol serum levels were the lowest. As calcidiol serum levels rose to the normal range in the course of the study, peritoneal losses of this metabolite increased slightly and correlated with calcidiol serum levels and urea mass transfer coefficient (MTC); the significant correlation between calcidiol serum levels and peritoneal protein losses disappeared. CONCLUSIONS: When serum calcidiol levels are low, calcidiol peritoneal losses in patients on CAPD correlate with protein peritoneal losses. However, when serum calcidiol levels rise, the calcidiol peritoneal losses correlate with calcidiol serum levels and urea MTC, and not with protein peritoneal losses.

Relations between calcidiol serum levels and bone mineral density in postmenopausal women with low bone density.

The relationship between vitamin D and bone density was studied in 150 selected, mature (45-74), postmenopausal women with a lumbar spine Z score below 0. Vitamin D status was evaluated using calcidiol serum levels. Serum calcitriol and parathyroid hormone (PTH) values were also evaluated in some subjects. Bone mass was evaluated by ascertaining bone density and Z and T scores in the lumbar spine and femur region. The reference group consisted of 25 premenopausal women. The postmenopausal group was divided into subgroups according to age, i.e., under or over 60 years old. Additionally, the whole group was also subdivided according to their lumbar spine Z scores into group I (Z > -1), group II (Z < -1; > -2), and group III (Z < -2). Group III of postmenopausal women had higher PTH and lower calcitriol levels than premenopausal women. Calcidiol serum levels were lower in postmenopausal women groups II or III than in the group I and premenopausal women. Calcidiol serum levels and the bone mass values for the lumbar spine were correlated positively in all the postmenopausal women; in the women over 60 years of age, calcidiol levels also correlated with the bone mass values expressed as the bone density in three femur regions: femoral neck, trocanter, and Ward's triangle. In conclusion, mature post-menopausal woman showed high PTH levels and low calcidiol and calcitriol values. Calcidiol status is significantly related to bone mineral density in the lumbar spine and in women over 60 years, calcidiol levels also correlated with bone density in the femur regions.

Fluorimetric determination of alkaline phosphatase activity in human serum by use of a flow-through biosensor.

A fluorimetric method for the determination of alkaline phosphatase activity based on the use of a flow-through biosensor is reported. The biochemical basis of the method is the hydrolysis of 4-methyl-umbelliferone-phosphate catalyzed by the analyte with fluorimetric monitoring of the 4-methyl-umbelliferone formed (lambda ex = 365 nm, lambda em = 445 nm). The enhancement of sensitivity achieved when the reaction product is retained on the support packed in the flow-cell makes the method suitable for determination of the analytes in serum samples after 1:50 dilution. The linear range is found to be between 0.1 and 20 U l-1, with relative standard deviation less than 2.2%. The use of this biosensor was tested by the determination of the analyte in human serum from healthy and sick individuals with excellent recoveries (94-103%).

Relation of abnormal composition of lipoproteins to HbA1c levels in non-insulin dependent diabetes.

Plasma lipids and VLDL and HDL composition were studied in a control group of 20 non diabetic subjects and in 31 male middle-age patients with non-insulin dependent diabetes treated by oral hypoglycemic agent glibenclamide and a weight maintaining diet. Data for the diabetics were separated based on haemoglobin A1c of less or greater than 7%. VLDL composition abnormalities were more frequent in the diabetic patients with HbA1c of > 7%. VLDL-cholesterol, VLDL-triglycerides and VLDL-phospholipids were high in all diabetics whereas VLDL-apo B increased only in diabetics with HbA1c > 7%. Apo CII and apo CIII levels and also apo CII/apo CIII ratio were also reduced in the diabetic patients with HbA1c levels of more than 7%. Increases in the apo E and apo E/apo C ratio were also seen in the more hyperglycemic diabetics with HbA1c levels > 7%. In contrast apo CII and apo CIII levels and also Apo CII/Apo CIII ratio remained unaltered in diabetic patients with less than 7% HbA1c levels. In these patients increases in the apo E levels were found while the apo E/apo C ratio remained unaltered. All diabetic patients showed increases in HDL-triglycerides and triglyceride/total cholesterol ratio with respect to control. Decreases in HDL-apo AI were also seen in both groups of diabetics, but the HDL-apo AI/HDL-apo AII ratio did not differ from control.

Serum osteocalcin concentrations in diabetic pregnant women and their newborns.

Osteocalcin and PTH serum levels were measured in 41 insulin-dependent diabetic pregnant women through the three trimesters of pregnancy with a total of 106 determinations of osteocalcin and 137 of PTH. In parallel we quantified these parameters in 90 normal pregnant women throughout the three trimesters of pregnancy. In addition calcitriol, osteocalcin and PTH levels were quantified at delivery in 16 diabetic pregnant women and 16 normal pregnant women at delivery, in cord serum and in the infants during the first days of life. Non-pregnant women (n = 48) were the control group. In normal pregnant women PTH levels increased during the third trimester and total calcitriol increased at delivery. Osteocalcin levels decreased in the second trimester but returned to normal values during the third trimester of pregnancy. Diabetic pregnant women showed constant PTH levels throughout pregnancy. At delivery in diabetic pregnant women, total calcitriol levels increased to a smaller extent than in normal pregnant women. Osteocalcin concentrations in the second and third trimester of pregnancy were lower than in the non-pregnant group. Infants of diabetic mothers showed lower PTH and osteocalcin concentrations than infants of normal pregnant women, whereas their calcitriol levels were similar. These data indicate that diabetes decreases bone turnover during pregnancy in the mother and during the perinatal period in their offspring.

Evidence of absorptive hypercalciuria in tuberculosis patients.

In patients with granulomatous diseases, disturbances in calcium metabolism have been described. The aim of the study was to evaluate alterations in calcium metabolism in patients with tuberculosis. Forty patients with tuberculosis (TB) were studied in a baseline state (calcium intake 1000 mg/day). Fourteen of these patients were also studied after restrictive calcium diet (400 mg/calcium/day) and after a load of oral calcium of 1000 mg. In all the studies, calcium and phosphorus were measured in serum and urine, and parathyroid hormone (PTH) in plasma. In addition, serum 25OHD and 1,25(OH)2D (calcitriol) levels were measured in the baseline state and after the restrictive diet. In the baseline state, 25OHD levels were lower and urinary calcium higher in TB patients than in the control group. No patients had hypercalcemia, but hypercalciuria was present in 10 patients (25%). The patients with tuberculosis were divided according to the presence or absence of hypercalciuria. In both groups, the 25OHD levels were lower than in controls. Hypercalciuric patients had lower plasma parathyroid hormone levels and higher serum calcitriol levels than the control group and the TB patients without hypercalciuria. Urinary calcium excretion after a calcium load was higher in TB patients with hypercalciuria than in controls. A positive correlation was found between the calcitriol levels and postcalcium load urinary calcium excretion in patients with calcium hyperabsorption. These data indicate that absorptive hypercalciuria is frequently observed in patients with TB and is possible due to inappropriately high serum calcitriol levels.