Ultrastructure of quick-frozen and freeze-substituted chick osteoclasts

For comparison with chemically fixed osteoclasts, we prepared chick osteoclasts by quick freezing followed by freeze-substitution. In spite of technical difficulties this demonstrated that osteoclasts can be satisfactorily frozen in situ by the metal contact method. Ultrastructural differences were revealed between conventional fixation and quick freezing. Compared with conventional fixation, the quick freezing method appeared to improve preservation: (1) a discrete trilaminar plasma membrane and other intracellular membranes showed a smooth profile without undulation or rupture; (2) cytoskeletal components appeared to be clearer, straighter, and more numerous; (3) the interior of the ruffled finger contained interconnected lattice structures whereas highly organised microfilaments were seen in the clear zone; (4) well developed tubulovesicular structures (TVSs) that branched or anastomosed with each other were revealed in the cytoplasm; (5) the contents of intracellular membrane systems including the nuclear envelope, endoplasmic reticulum, and Golgi complex were stained to a various extent; (6) vesicles and vacuoles were much smaller, round and well-defined with electron-dense contents; (7) crystalline structures were seen at the extracellular channels of the ruffled border, in the lumen of TVSs, and in vesicles; (8) in some instances mitochondrial granules were visible; (9) within the resorptive lacuna, osteoclasts adhered to the degraded bone matrix without any intervening empty space.     

Urinary pregnanetriol-3-glucuronide in children: Age-related change and application to the management of 21-hydroxylase deficiency

Urinary concentrations of pregnanetriol-3-glucuronide (PT-3-G) were determined in 485 normal Japanese subjects (277 males and 208 females), aged 5 days to 20 years, using an enzyme-linked immunosorbent assay (ELISA). The usefulness of urinary PT-3-G concentrations before giving the morning dose of medications in monitoring the adequacy of glucocorticoid treatment was assessed in eight patients with 21-hydroxylase deficiency (21-OHD). The ratio of PT-3-G to excreted creatinine (PT-3-G/Cre ratio) increased significantly during the first month and did not change from age 1 month to 1 year of life. The ratio decreased to a nadir at age 3 or 4 years followed by continuous, significant increase until late adolescence. In the subjects treated with corticosteroids for 21-OHD, PT-3-G/Cre ratios at the 50th percentile or below suggested a risk of excessive treatment, as judged by the patients' growth. Measurement of the PT-3-G/Cre ratio enabled recognition of corticosteroid overtreatment, which was not demonstrated by determining the serum concentrations of 17α-hydroxyprogesterone (17-OHP) On the other hand, ratios at the upper 95–99% tolerance limits seemed to be required for optimal control. The present study revealed the normal age-related changes in urinary excretion of PT-3-G and showed it to be a reliable marker for evaluating glucocorticoid treatment in young children with 21-OHD.