Microwave-assisted derivatization: application to steroid profiling by gas chromatography/mass spectrometry.

Gas chromatography-mass spectrometry (GC-MS) remains as the gold-standard technique for the study of the steroid metabolome. A main limitation is the need of performing a derivatization step since incubation with strong silylations agents for long periods of time (usually 16 h) is required for the derivatization of hindered hydroxyls present in some steroids of interest. In the present work, a rapid, simple and reproducible microwave-assisted derivatization method was developed. In the method, 36 steroids already treated with methoxyamine (2% in pyridine) were silylated with 50 µl of N-trimethylsilylimidazole by using microwave irradiation, and the formed methyloxime-trimethylsilyl derivatives were analyzed by GC-MS. Microwave power and derivatization time silylation conditions were optimized being the optimum conditions 600 W and 3 min respectively. In order to evaluate the usefulness of this technique, the urine steroid profiles for 20 healthy individuals were analyzed. The results of a comparison of microwave irradiation with the classical heating protocol showed similar derivatization yields, thus suggesting that microwave-assisted silylation is a valid tool for the rapid steroid metabolome study.

Utilidad clínica de la determinación del factor de crecimiento de los fibroblastos 23 en la valoración de pacientes con osteomalacia / Clinical usefulness of the determination of fibroblast growth factor 23 in the evaluation of patients with osteomalacia

Fundamento y objetivo: El objetivo de este estudio ha sido analizar la utilidad de la determinación del fibroblast growth factor 23 (FGF23, «factor de crecimiento de los fibroblastos 23»), una hormona reguladora del metabolismo del fosfato, en la valoración de pacientes con osteomalacia de distintas causas. Pacientes y método: Se incluyeron 17 pacientes con osteomalacia: 12 hipofosfatémica (de distintas causas), 4 por déficit de vitamina D y uno por hipofosfatasia. En todos ellos se determinó el FGF23 C -terminal plasmático. Resultados: Se observó un aumento del FGF23 en 6/12 (50%) pacientes con osteomalacia hipofosfatémica (2 ligada a cromosoma X , una autosómica dominante, una asociada a tratamiento por VIH y 2 no filiadas). Ningún paciente con osteomalacia por déficit de vitamina D o hipofosfatasia presentó un aumento del FGF23. Conclusión: La determinación del FGF23 puede ser útil en la valoración de los distintos tipos de osteomalacia hipofosfatémica y en la identificación del mecanismo etiopatogénico asociado. Así, el 50% de los pacientes con osteomalacia hipofosfatémica, dependiendo de su etiología, tienen un aumento de FGF23, mientras que en la osteomalacia por déficit de vitamina D y en la hipofosfatasia los valores de esta hormona son normales

Background and objective The aim of the present study was to analyze the usefulness of the determination of fibroblast growth factor 23 (FGF23), a regulatory hormone of phosphate metabolism, in the evaluation of patients with osteomalacia of different causes. Patients and method Seventeen patients with osteomalacia were included: 12 hypophosphatemic osteomalacia (by several causes), 4 vitamin D-deficiency osteomalacia and one with hypophosphatasia. Plasma C-terminal FGF23 was determined in all patients. Results FGF23 levels were increased in 6/12 (50%) of patients with hypophosphatemic osteomalacia (2 X -linked, one autosomal dominant, one related HIV therapy and 2 not elucidated). No patient with vitamin D-deficiency osteomalacia or hypophosphatasia presented increased FGF23 levels. Conclusion The determination of FGF23 could be useful in the evaluation of the different types of hypophosphatemic osteomalacia and also in the identification of their associated etiopathogenic mechanisms. Thus, depending on the cause, 50% of the patients with hypophosphatemic osteomalacia showed increased FGF23 values, whereas in vitamin D-deficiency osteomalacia and in hypophosphatasia FGF23 levels were normal (AU)

[Clinical usefulness of the determination of fibroblast growth factor 23 in the evaluation of patients with osteomalacia]. / Utilidad clínica de la determinación del factor de crecimiento de los fibroblastos 23 en la valoración de pacientes con osteomalacia.

BACKGROUND AND OBJECTIVE: The aim of the present study was to analyze the usefulness of the determination of fibroblast growth factor 23 (FGF23), a regulatory hormone of phosphate metabolism, in the evaluation of patients with osteomalacia of different causes. PATIENTS AND METHOD: Seventeen patients with osteomalacia were included: 12 hypophosphatemic osteomalacia (by several causes), 4 vitamin D-deficiency osteomalacia and one with hypophosphatasia. Plasma C-terminal FGF23 was determined in all patients. RESULTS: FGF23 levels were increased in 6/12 (50%) of patients with hypophosphatemic osteomalacia (2 X-linked, one autosomal dominant, one related HIV therapy and 2 not elucidated). No patient with vitamin D-deficiency osteomalacia or hypophosphatasia presented increased FGF23 levels. CONCLUSION: The determination of FGF23 could be useful in the evaluation of the different types of hypophosphatemic osteomalacia and also in the identification of their associated etiopathogenic mechanisms. Thus, depending on the cause, 50% of the patients with hypophosphatemic osteomalacia showed increased FGF23 values, whereas in vitamin D-deficiency osteomalacia and in hypophosphatasia FGF23 levels were normal.

Effects of bilirubin and sera from jaundiced patients on osteoblasts: contribution to the development of osteoporosis in liver diseases.

UNLABELLED: Low bone formation is considered to be the main feature in osteoporosis associated with cholestatic and end-stage liver diseases, although the consequences of retained substances in chronic cholestasis on bone cells have scarcely been studied. Therefore, we analyzed the effects of bilirubin and serum from jaundiced patients on viability, differentiation, mineralization, and gene expression in the cells involved in bone formation. The experiments were performed in human primary osteoblasts and SAOS-2 human osteosarcoma cells. Unconjugated bilirubin or serum from jaundiced patients resulted in a dose-dependent decrease in osteoblast viability. Concentrations of bilirubin or jaundiced serum without effects on cell survival significantly diminished osteoblast differentiation. Mineralization was significantly reduced by exposure to 50 µM bilirubin at all time points (from -32% to -55%) and jaundiced sera resulted in a significant decrease on cell mineralization as well. Furthermore, bilirubin down-regulated RUNX2 (runt-related transcription factor 2) gene expression, a basic osteogenic factor involved in osteoblast differentiation, and serum from jaundiced patients significantly up-regulated the RANKL/OPG (receptor activator of nuclear factor-κB ligand/osteoprotegerin) gene expression ratio, a system closely involved in osteoblast-induced osteoclastogenesis. CONCLUSION: Besides decreased cell viability, unconjugated bilirubin and serum from jaundiced patients led to defective consequences on osteoblasts. Moreover, jaundiced serum up-regulates the system involved in osteoblast-induced osteoclastogenesis. These results support the deleterious consequences of increased bilirubin in advanced chronic cholestasis and in end-stage liver diseases, resulting in disturbed bone formation related to osteoblast dysfunction.

Osteomalacia revisited : a report on 28 cases.

The aim of this study was to analyse the clinical manifestations and the most frequent causes of osteomalacia (OM) in a group of 28 patients diagnosed with this disorder during a 20-year period. OM was diagnosed by bone biopsy and/or by Bingham and Fitzpatrick criteria (two of the following: low calcium, low phosphate, elevated total alkaline phosphatase [total AP] or suggestive radiographs). Of these patients, 13 had vitamin D deficiency OM (VD-OM), 14 hypophosphatemic OM (HypoP-OM) and one had OM-associated hypophosphatasia. Deficient sun exposure and celiac disease were the most frequent etiologies of VD-OM, whereas most HypoP-OM were hereditary forms. The main clinical symptoms were polyarthralgias (89%), frequently associated with fractures (75%). Fifty seven percent had densitometric criteria of osteoporosis. Patients with VD-OM showed significantly higher total AP and PTH serum values, but lower vitamin D, serum calcium, calciuria and bone mass than patients with HypoP-OM. Conversely, HypoP-OM patients had significantly lower serum phosphate and higher phosphaturia than patients with VD-OM. Briefly, high total AP, low serum calcium and low serum phosphate were observed in 85%, 65% and 15%, respectively, of patients with VD-OM, being observed in 64%, 14% and 100%, respectively, of HypoP-OM patients. Nearly 50% of these latter showed increased FGF23 levels. In conclusion, in this study, the frequencies of HypoP-OM and VD-OM were similar. The most frequent laboratory abnormalities were increased total AP and decreased serum phosphate. A urinary calcium loss of less than 50 mg/dl was highly discriminatory for VD-OM and a serum phosphate less than 2.3 mg/dl was also high discriminatory for HypoP-OM. Low densitometric values and fractures were frequent among these patients.

Use of gonadotropin-releasing hormone agonists in patients with Hodgkin's disease for preservation of ovarian function and reduction of gonadotoxicity related to chemotherapy.

In young women receiving chemotherapy for Hodgkin's disease, the combined use of triptorelin and tibolone cotreatment may be a useful tool for preserving ovarian function because all but three (10%) of the women in this treatment group returned to spontaneous ovulation and menses, in contrast to 23% of subjects in the control group (P<.05). No significant differences were observed in bone mineral density between groups.

Clinical, biochemical and morphologic diagnostic markers in an infant male pseudohermaphrodite patient with compound heterozygous mutations (G115D/R246W) in SRD5A2 gene.

A patient with male pseudohermaphroditism and clinical diagnosis of partial androgen insensitivity in the neonatal period was studied at pubertal age for a molecular diagnosis. Hormone studies were conducted at baseline and under hCG stimulation for testosterone and dihydrotestosterone determinations at 2 months of age. Gonadectomy was performed at 4 months. At the age of 13 years genital skin fibroblasts were studied for androgen binding and 5alpha-reductase activity and peripheral blood DNA was available for androgen receptor (AR) and 5alpha-reductase (SRD5A2) gene analysis. Exons 1-8 of AR gene and exons 1-5 of SRD5A2 gene were sequenced. AR gene coding sequences were normal. SRD5A2 gene analysis revealed two heterozygote mutations (G115D and R246W), with the mother carrying the G115D and the father the R246W mutations. The compound heterozygote mutations in SRD5A2 gene explained an extremely low 5alpha-reductase enzyme activity in genital skin fibroblasts. Revision of hormonal data from the neonatal period revealed an increased testosterone-to-dihydrotestosterone ratio at the end of an hCG stimulation test, which concurred with the molecular diagnosis. Testis morphology at 4 months of age was normal. Clinical and biochemical differential diagnosis between partial androgen insensitivity syndrome and 5alpha-reductase enzyme deficiency is difficult in the neonatal period and before puberty. Our results show that in our patient the testosterone-to-dihydrotestosterone ratio would have adequately orientated the diagnosis. The two mutations in SRD5A2 gene have been described in patients of different lineages, though not in combination to date. Testis morphology showed that, during early infancy, the 5alpha-reductase deficiency may not have affected interstitial or tubular development.

Effect of hemodialysis and renal failure on serum biochemical markers of bone turnover.

The aim of the present study was to evaluate the effect of hemodialysis and renal failure on serum bone markers. Serum total alkaline phosphatase (TAP), procollagen type I aminoterminal propeptide (PINP), and beta-carboxyterminal telopeptide of type I collagen (beta-CTX), as well as intact parathyroid hormone (iPTH), creatinine, and total protein were measured in 14 patients with endstage renal disease (ESRD) before and at 1, 2, and 4 h during a hemodialysis session, and at the same sampling interval in 6 renal transplant recipients. The results were compared to those obtained in 20 healthy adults. All patients showed increased baseline mean values of PINP, beta-CTX, and iPTH. Beta-CTX differed significantly between hemodialysis patients and renal transplant recipients. TAP and beta-CTX were the only markers which correlated with iPTH ( P << 0.05) and creatinine values ( P << 0.001), respectively. Renal transplant recipients did not show significant variations in the evolution of mean values of bone markers throughout the study, whereas, during the dialysis period, all the bone markers analyzed in the study showed a significant change. The change differed depending on the marker considered: beta-CTX showed a significant decrease at the end of the session, TAP increased at this time and, although PINP showed an initial increase during hemodialysis, no significant changes were observed at the end of the session. We conclude that bone markers are significantly influenced by hemodialysis, especially serum TAP and beta-CTX. ESRD is associated with an increase in these bone markers, in some cases related to iPTH values and in others to glomerular function. These findings should be taken into account when evaluating bone markers in these patients.

Secreting ovarian tumors may protect women from osteoporosis.

OBJECTIVE: It is well known that ovarian steroids modulate bone turnover. Conditions associated with low levels of these hormones, such as menopause, hypogonadism, and others, have been related to osteopenia or osteoporosis. On the other hand, hyperandrogenism in premenopausal women, mainly in polycystic ovarian syndrome, has been reported to have a protective effect on bone mass. However, data regarding how bone mass is affected by neoformative processes in which steroids are increased are not as well documented. Our aim was to study the effect of secreting ovarian tumors on bone mass. METHODS: A total of 14 patients were referred to our hospital because of endocrine ovarian tumors. Steroid levels were measured prior to and after surgery. Bone mineral density (BMD) by DEXA was assessed at inclusion in all cases. Additionally, in 7 women bone measurement was repeated after 1-year follow-up. The setting was a tertiary hospital. RESULTS: All patients showed increased levels of testosterone, androstenedione, and free testosterone prior to surgery. BMD was also in the normal-upper range or over normal in all of them. As expected in the subjects with a second DEXA a decrease in bone mass was noted. CONCLUSION: Steroid secreting ovarian tumors increase bone mass and thus may protect women from later osteoporosis.