Reduced skin thickness: a new minor diagnostic criterion for the classical and hypermobility types of Ehlers-Danlos syndrome.

BACKGROUND: The diagnosis of Ehlers-Danlos syndrome (EDS) is mainly based on clinical criteria, although in some instances a sound molecular diagnosis is available. Clinical signs can be divided into two categories: one with high diagnostic specificity and the other with low specificity. Despite the fact that reduced skin thickness is one of the dermatological features in patients with EDS, this issue has not been analysed in greater detail. OBJECTIVES: To determine skin thickness in patients with the classical and the hypermobility types of EDS. METHODS: In 21 patients with classical type of EDS and in nine patients with hypermobility type of EDS, skin thickness was analysed at different body sites by cross-sectional b-mode scans obtained with a 20-MHz ultrasound system. RESULTS: We found a significant decrease in skin thickness in both types of EDS, which was highest at the chest and at the distal part of the lower leg. CONCLUSIONS: We propose that the reduced thickness of the dermis as determined by high-resolution 20-MHz ultrasound can be used as a new minor criterion in the diagnosis of the classical and the hypermobility types of EDS.

Interactions of fibrillin-1 with heparin/heparan sulfate, implications for microfibrillar assembly.

Fibrillin-1 is a major constituent of the 10-12 nm extracellular microfibrils. Here we identify, characterize, and localize heparin/heparan sulfate-binding sites in fibrillin-1 and report on the role of such glycosaminoglycans in the assembly of fibrillin-1. By using different binding assays, we localize two calcium-independent heparin-binding sites to the N-terminal (Arg(45)-Thr(450)) and C-terminal (Asp(1528)-Arg(2731)) domains of fibrillin-1. A calcium-dependent-binding site was localized to the central (Asp(1028)-Thr(1486)) region of fibrillin-1. Heparin binding to these sites can be inhibited by a highly sulfated and iduronated form of heparan sulfate but not by chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate, demonstrating that the heparin binding regions represent binding domains for heparan sulfate. When heparin or heparan sulfate was added to cultures of skin fibroblasts, the assembly of fibrillin-1 into a microfibrillar network was significantly reduced. Western blot analysis demonstrated that this effect was not due to a reduced amount of fibrillin-1 secreted into the culture medium. Inhibition of the attachment of glycosaminoglycans to core proteins of proteoglycans by beta-d-xylosides resulted in a significant reduction of the fibrillin-1 network. These studies suggest that binding of fibrillin-1 to proteoglycan-associated heparan sulfate chains is an important step in the assembly of microfibrils.

Age-related differences in the expression of receptors for TGF-beta in human osteoblast-like cells in vitro.

The cell surface expression of receptors for TGF-beta was studied in human osteoblasts derived from femoral trabecular bone of a total of 19 patients aged 2-83 years. All cell populations investigated showed a similar profile of expression of TGF-beta receptors (TbetaR) I, II and III (betaglycan). There were no significant differences in cell differentiation or proliferative behaviour between the age groups. The TGF-beta receptor number per cell significantly increased with age, while the receptor affinity tended to decrease. IGF-I did not influence TbetaR expression in vitro. The results indicate an age-dependent and IGF-I independent increase of osteoblastic TGF-beta receptors in human osteoblast-like cells in vitro.

Insulin activation of insulin receptor kinase in erythrocytes is not altered in non-insulin-dependent diabetes and not influenced by hyperglycemia.

Recent studies suggest that high glucose concentrations impair insulin receptor phosphorylation and kinase activation in certain cell models. To examine whether such an effect of glucose can also be demonstrated in vivo, insulin receptor kinase activation was studied in erythrocytes from 11 patients with non-insulin-dependent diabetes (NIDDM), before and after reduction of hyperglycemia (from 14.6+/-1.6 to 6.6+/-0.5 mmol/l fasting plasma glucose within 8.6+/-0.6 days). For the measurement of receptor kinase activation, cells were incubated with insulin (0-400 nmol/l), solubilized and insulin receptors immobilized to microwells coated with anti-insulin receptor antibody. Kinase activity towards insulin receptor substrate-1 and insulin binding were then measured in these wells. Kinase activities (expressed as amol phosphate transferred per min and per fmol insulin binding activity) were similar before (2.4+/-0.4 and 32.2+/-2.0 amol/min per fmol with 0 and 400 nmol/l insulin, respectively) and after improvement of metabolic control (2.4+/-0.5 and 32.0+/-2.3 amol/min per fmol with 0 and 400 nmol/l insulin, respectively). Moreover, activities were also similar in 22 hyperglycemic patients with NIDDM (2.1+/-0.3 and 35.1+/-1.4 amol/min per fmol with 0 and 400 nmol/l insulin, respectively) compared with those in 21 non-diabetic control individuals (2.1+/-0.3 and 34.2+/-1.2 amol/min per fmol with 0 and 400 nmol/l insulin, respectively). We conclude that insulin activation of erythrocyte insulin receptor kinase is not impaired in NIDDM and is not influenced by hyperglycemia.

[Severe vitamin D deficiency osteomalacia by coincidence of multiple risk factors]. / Schwere Vitamin-D-Mangel-Osteomalazie durch Koinzidenz mehrerer Risikofaktoren.

HISTORY: An 82-year-old woman was admitted with bilateral clavicular fractures after falling out of bed. She complained of pain in all her bones. She reported having undergone a partial gastrectomy (Billroth II) 40 years ago with subsequent milk intolerance and a dislike of direct sun light. INVESTIGATIONS: Scintigraphy revealed multiple areas of abnormal uptake. Conventional radiography showed in both ulnae transverse zones of increased density with a central translucent band. Laboratory tests demonstrated the constellation of increased bone turnover in secondary hyperparathyroidism with hypocalcaemia (1.49 mmol/l) and hypophyosphataemia (0.62 mmol/l). The 25-hydroxyvitamin D-level was too low to be measured. Ileal crest biopsy indicated osteomalacia. TREATMENT AND COURSE: Serum levels of calcium, alkaline phosphatase and vitamin D became normal after 7 weeks of daily administration of 10,000 IU vitamin D and 1000 mg calcium. The severe pains had subsided after two weeks and no longer required analgesics. CONCLUSION: Multiple areas of increased uptake on skeletal scintigraphy, combined with diffuse bone pain and increased alkaline phosphatase, were at first misinterpreted as extensive skeletal metastases. But the history, at first seemingly of little significance, contained three long-term risk factors for the development of vitamin D and calcium deficiency and thus of osteomalacia, namely partial gastrectomy, milk intolerance and inadequate exposure to sun light. Early recognition of risk factors for bone changes will rapidly lead to the correct diagnosis and immediate treatment of the treatable underlying disease.

Increased cell surface expression of receptors for transforming growth factor-beta on osteoblasts from patients with Osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a heritable connective tissue disorder usually characterized by either a reduction in the production of normal collagen I or the synthesis of abnormal collagen. The variability in the clinical phenotype is not in each case sufficiently explained by the underlying mutation in the collagen I genes. Also, biochemical differences between mutant collagen from different tissues suggest additional regulatory mechanisms possibly involved in matrix deposition and maturation, two processes in which transforming growth factor-beta (TGF-beta) plays an important role. We, therefore, studied the cell surface expression and functional properties of TGF-beta receptors I, II and III on osteoblasts from a group of OI patients compared to healthy controls. Receptor number and affinity were determined by Scatchard analysis of binding data and TGF-beta receptor II gene expression was assessed by RT-PCR. Ligand-induced downregulation of TGF-beta receptors was analyzed to demonstrate the dynamic response to exogenous stimuli. All experiments were performed in parallel in human osteoblastic cells from OI patients and from age-matched controls. TGF-beta receptors I, II and III (betaglycan) were present on osteoblasts from both healthy donors and OI patients. The receptor numbers were significantly higher (29,000 per cell) on OI osteoblasts than on age-matched control osteoblasts (12,000 per cell) in spite of similar steady state levels for TGF-beta receptor II mRNA in OI and control cells. Furthermore, receptor affinity was not significantly different in OI osteoblasts (181 vs. 177 nM(-1)), and the receptor number did not depend on the culture substrate. With respect to dynamic adaption, ligand-induced downregulation of TGF-beta receptors was reduced in OI osteoblasts. In conclusion, the human osteoblastic cells from patients with OI investigated all have an elevated number of cell surface receptors for TGF-beta, without any evidence for a transcriptional regulation of TGF-beta receptor II. On the functional level, there is some evidence for an impaired adaptive behavior of receptor presentation, whereas receptor affinity is unchanged.

Overhydroxylation of lysyl residues is the initial step for altered collagen cross-links and fibril architecture in fibrotic skin.

In fibrotic skin of lipodermatosclerosis a substantial increase of the cross-link hydroxylysylpyridinoline is observed. Hydroxylysylpyridinoline is a typical cross-link of skeletal tissue and is thought to play a major part in the hardening of sclerotic tissue. We investigated whether the increase in hydroxylysylpyridinoline is due to overhydroxylation of lysyl residues in the collagen molecule, which may also be associated with an increase of glycosylated hydroxylysine residues. Furthermore, we determined whether the collagen fibrils in lipodermatosclerosis showed a decrease of the diameter in the tissue as well as in vitro after fibrillogenesis of pepsin-solubilized collagens. Isolated alpha-chains of pepsin solubilized collagen I showed an increase in lysyl hydroxylation (hyl/(hyl + lys)) as compared with normal control [alpha1(I): lipodermatosclerosis 0.18 +/- 0.01; control 0.12 +/- 0.01; alpha2(I): lipodermatosclerosis 0.36 +/- 0.02; control 0. 25 +/- 0.03, p < 0.001]. Furthermore, the content of enzymatic glycosylated hydroxlysine residues increased. This increase is associated with a decrease of fibril diameter of both tissue and fibrils formed in vitro of pepsin-solubilized collagens. In the same pool of collagens an increase in collagen III content was observed as compared with controls (lipodermatosclerosis 14.5% +/- 1.6, control 10.3% +/- 1.6, p < 0.001). Our results showed that the overhydroxylation of lysyl residues, which is required for the generation of hydroxylysylpyridinoline, is not only restricted to the telopeptides but also affects the helical part of the molecule. This process is further associated with an increase of glycosylated hydroxylysyl residues. These changes along with the increase in collagen III content seem to be responsible for the observed alteration in the architecture of collagen fibrils in sclerotic skin.

Hypergravity stimulates collagen synthesis in human osteoblast-like cells: evidence for the involvement of p44/42 MAP-kinases (ERK 1/2).

The formation and organization of skeletal tissue is strongly influenced by mechanical stimulation. There is increasing evidence that gravitational stress has an impact on the expression of early response genes in mammalian cells and may play a role in the formation of extracellular matrix. In particular, osteoblasts may be unique in their response to gravitational stimuli since in these cells microgravity has been reported to reduce collagen synthesis, while in fibroblasts the opposite effect was observed. Here, we have investigated the influence of hypergravity induced by centrifugation on the collagen synthesis of human osteoblast-like cells (hOB) and studied the possible involvement of the mitogen-activated protein (MAP) kinase signaling cascade. Collagen synthesis was significantly increased by 42+/-16% under hypergravity at 13 x g, an effect paralleled by the enhanced expression of the collagen I alpha 2 (COL1A2) mRNA. No difference was seen in the proportion of collagen types I, III, and V synthesized by hOB. Hypergravity induced a markedly elevated phosphorylation of the p44/42 MAP kinases (ERK 1/2). The inhibition of this pathway suppressed the hypergravity-induced stimulation of both collagen synthesis as well as COL1A2 mRNA expression by about 50%. Our results show that the collagen synthesis of non-transformed hOB is stimulated under hypergravitational conditions. This response appears to be partially mediated by the MAP kinase pathway.

[Ehlers-Danlos syndrome]. / Ehlers-Danlos-Syndrom.

The Ehlers-Danlos syndrome (EDS) comprises a heterogenous group of nine hereditary connective tissue disorders, characterized by hyperelasticity of skin and hypermobility of joints to differing extents. The skin is easily injured and wound healing is delayed. The majority of EDS patients belong to EDS-types I-III. The pathogenesis in these cases is not known, although recent data suggest a role for collagen V. In contrast, the etiology of EDS-types IV, VI and VII has been found. While EDS IV is caused by a mutation in the collagen III gene, in EDS VI a mutation in the lysyl hydroxylase gene is present. In EDS VII, the underlying defect is a mutation in the collagen I gene. The EDS-types V, VII and X are very rare; their symptoms resemble those of EDS-type II.

Ligand-induced downregulation of receptors for TGF-beta in human osteoblast-like cells from adult donors.

High concentrations of transforming growth factor b (TGF-beta) are found in the bone matrix, reflecting a pivotal role of this growth factor in the coupling of bone resorption and formation. TGF-beta strongly stimulates the synthesis of extracellular matrix proteins, but in vitro studies show an inhibitory effect on the final mineralization process, which in vivo occurs despite high concentrations of TGF-beta. Little is known about how bone-forming cells respond to different concentrations of TGF-beta and if they can transiently adapt receptor numbers in order to modulate cellular activity. Against this background, we studied the cell-surface expression of TGF-beta receptors (TbetaR) I, II and III (betaglycan) on human osteoblast-like cells from adult donors, and examined the TbetaR presentation on these cells after a preceding exposure to TGF-beta1. Affinity crosslinking studies with disuccinimidylsuberate showed the presence of all three receptor types. Preincubation with TGF-beta1 markedly reduced 125I-TGF-beta1 binding in a time-dependent and dose-dependent manner and revealed a 95% reduction after an 18-h preincubation with 200 pM TGF-beta1. In parallel, Scatchard analysis showed that the binding affinity did not change as a consequence of TGF-beta1 preincubation. Immunoblotting analyses revealed an almost complete disappearance of immunoreactive TbetaR-II and TbetaR-III proteins after a 24-h preincubation with TGF-beta1. Using semi-quantitative reverse transcription PCR, no effect of TGF-beta1 on the expression of TbetaR-II mRNA was observed. These studies demonstrate a ligand-induced downregulation of TbetaRs-II and -III on human osteoblast-like cells, without any evidence for recovery within the first 24 h, both in the presence and after the removal of the ligand. The underlying mechanism appears to be based on post-transcriptional events. The results suggest that high concentrations of active TGF-beta1 decrease the responsiveness of osteoblasts towards this growth factor.

[Pseudohyperthyroxinemia in endemic sprue--reversibility with a gluten-free diet]. / Pseudohyperthyroxinämie bei einheimischer Sprue--Reversibilität unter glutenfreier Diät.

HISTORY: An increased concentration of free T3 (fT3) with normal TSH was repeatedly measured in a 58-year-old man with diarrhoea. Hyperthyroidism was diagnosed and he was treated with thyrostatic drugs. INVESTIGATIONS: Referred to our clinic, fT4 concentration, determined by routine ELISA, was found to be raised to 33.9 pmol/l, while fT3 and TSH levels were within normal limits. But the equilibrium-dialysis method gave a normal serum level also for fT4. Gastroscopy suggested coeliac disease, confirmed histologically. TREATMENT AND COURSE: The symptoms quickly improved on a gluten-free diet and the fT4 level returned to normal values even on routine measurement. CONCLUSION: The raised fT4 level with non-suppressed TSH was only revealed as a false finding by special non-routine laboratory methods. The reversibility and the close time relationship of the reported laboratory data with the clinical manifestations of the concomitant coeliac disease strongly suggested that changes in serum in the course of the latter disease played a role in these misleading laboratory results. An interference of coeliac disease with some laboratory tests has been previously described.

Glycosylation of human bone collagen I in relation to lysylhydroxylation and fibril diameter.

Posttranslational modifications (lysylhydroxylation, glycosylation, and crosslink formation) of collagen I and the trabecular bone volume (TBV) as well as the supramolecular organization of human vertebrae were studied by analyzing vertebral bones of 55 individuals (22-93 years of age). The degree of lysylhydroxylation of both a-chains of collagen I showed a significant inverse correlation with the TBV, while only a weak correlation between lysylhydroxylation of alpha2(I) and the age of the donor was observed. The degree of glycosylation of collagen I was significantly correlated with both the level of lysylhydroxylation and the degree of osteopenia. Electron microscopic evaluation did not show any relationship between the level of collagen glycosylation and the diameter of in vivo formed fibrils or in vitro formed fibrillar aggregates. In our study the molar ratio of the mature collagen crosslinks, pyridinoline and deoxypyridinoline, showed a slight tendency to be higher, in particular in the samples with a high level of lysylhydroxylation. This ratio was recently found to be significantly increased in avian osteoporotic bone. Our data suggest that the increased level of lysylhydroxylation in human osteopenia is related to the glycosylation of collagen I, while it seems to have little impact on the formation of the mature, non-reducible collagen crosslinks investigated. Based on our observations it appears unlikely that the different diameters of collagen fibrils contribute greatly to the reduced biomechanical stability reported for overhydroxylated, osteopenic bone tissue.

Semipreparative isolation of collagen types I, II, III and V by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroelution.

A simple method for the isolation of alpha-chains of different collagen types was developed. The procedure involves sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electroelution of separated and defixed collagen alpha-chains. Collagen types I, II, III and V from different porcine tissues were recovered in high quantity (> 95%) and purity (> 98%) as evidenced by amino acid analysis. The procedure can be used for sample quantities smaller than required for conventional methods e.g. chromatographic procedures.

Altered x-ray diffraction pattern is accompanied by a change in the mode of cross-link formation in lipodermatosclerosis.

We studied the molecular packing of collagen fibrils by x-ray diffraction in skin specimens of patients with lipodermatosclerosis and in controls. A difference in the tilt angles of the collagen molecules relative to the fiber axis is suggested by a D-stagger that is 1 nm larger in sclerotic skin than in normal skin. In parallel, the collagen cross-links in the skin specimens were analyzed, and a marked increase of both hydroxylysylpyridinoline and lysylpyridinoline, the trivalent mature cross-links characteristic of skeletal tissues, was found. The content of hydroxylysylpyridinoline and lysylpyridinoline was higher in the deep layer of the affected dermis than in the superficial dermis. This increase was always accompanied by an increase in the hydroxylysylpyridinoline/lysylpyridinoline ratio, suggesting that hydroxylysylpyridinoline is a sclerosis-associated cross-link. In addition, lysyl hydroxylation was increased in affected skin, and this increase was apparently restricted to the collagen telopeptides, which are crucial anchoring structures for lysyl dependent cross-links.

Changes with age in the urinary excretion of hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP).

There is increasing evidence that the measurement of urinary hydroxylysylpyridinoline (HP or PYD) and lysylpyridinoline (LP or DPD) by HPLC (high performance liquid chromatography) is potentially useful in clinical and pharmacological studies. HP and LP are promising markers of bone resorption because their levels in urine reflect the breakdown of mature collagen fibrils mainly of skeletal tissues. HP and LP are two non-reducible cross-links of mature collagen which are formed by a sequence of post-translational modifications. HP is a derivative of three residues of hydroxylysine and is present in almost all mature tissues (e.g. tendon. vessel walls, cartilage, dentine and bone). LP is a derivative of two residues of hydroxylysine and one residue of lysine and is present mainly in dentine and bone. Neither cross-link is found in normal human skin. We have isolated and purified HP and LP from commercially available bone gelatine by a preparative reverse-phase column HPLC. These two components were used as external standards for sample analysis. In the present study we analysed the urinary excretion of HP and LP in a group of 264 male and 279 female healthy subjects aged from 6 months to 65 years. A continuous decline of both cross-link components during childhood paralleled by a decrease of the HP:LP-ratio was observed. The levels of HP and LP were 2.5-5 times higher in infants (0.5-1 year) than in children (5-10 years) and 15-20 times higher than in adults (26-65 years). After the age of 17 years, both parameters remained at low levels. These data allow a precise quantitative monitoring of bone resorption in patients with metabolic bone diseases or during pharmacological interventions.

Microgravity and hypergravity effects on collagen biosynthesis of human dermal fibroblasts.

Astronauts experiencing long periods of space flight suffer from severe loss of bone tissue, particularly in those bones that carry the body weight under normal gravity. It is assumed that the lack of mechanical load decreases connective tissue biosynthesis in bone-forming cells. To test this assumption, quantitative and qualitative aspects of collagen synthesis under microgravity, normal gravity, and hypergravity conditions were investigated by incubating human fibroblast cultures with [3H]-proline for 4, 7, 10, and 20 h during the Spacelab D2-mission in 1993. Quantitative analysis revealed an increase of collagen synthesis under microgravity conditions, being up to 143% higher than in 1 g controls. In contrast, hypergravity samples showed a decrease in collagen synthesis with increasing g, being at the 13% level at 10 g. The relative proportion of collagen in total synthesized protein showed a slight decrease with increasing g. The secretion of collagen by the cells, proline hydroxylation of individual collagen alpha-chains, and the relative proportions of synthesized collagens I, III, and V were not affected under any of the applied conditions.

Transforming growth factor beta 1 influences lysyl hydroxylation of collagen I and reduces steady-state levels of lysyl hydroxylase mRNA in human osteoblast-like cells.

Transforming growth factor beta 1 (TGF-beta 1) is an osteotropic growth factor that is found in substantial concentration in bone. The authors studied the influence of TGF-beta 1 on the modification of lysine residues of collagen I. The degree of lysyl hydroxylation and lysyl glycosylation of newly synthesized collagen as well as steady-state levels of mRNA for both lysyl hydroxylase and collagens I and III were determined in human osteoblast-like cells in vitro. In normal human osteoblasts lysyl hydroxylation was decreased by TGF-beta 1 particularly in the collagen alpha 2-chain. This effect was paralleled by an increase in lysyl residues, whereas glycosylation was not affected. The mRNA for lysyl hydroxylase was reduced by one-third under the influence of TGF-beta 1. Additionally, the mRNAs for both procollagen I alpha-chains were stimulated by TGF-beta 1, whereas pro alpha 1 (III)-mRNA showed a decrease. Changes in the local regulatory activity of TGF-beta 1 may play a role in matrix maturation such as collagen type production and lysyl hydroxylation, the latter being altered in various pathological conditions, e.g. in generalized osteopenia.

[The clinical course and therapy of massive theophylline poisoning]. / Klinischer Verlauf und Therapie einer massiven Theophyllin-Intoxikation.

With suicidal intent a 72-year-old man swallowed 5.8 g aminophylline in a non-retard solution. The theophylline plasma level on admission was 120 mg/l. He had to be intubated when respiratory arrest occurred. Within the first hour he developed cerebral seizures, polymorphous ventricular premature systoles, atrial fibrillation with an irregular ventricular rate and, finally, recurrent episodes of ventricular fibrillation with prolonged circulatory shock (heart rate 120-140/min with a systolic blood pressure of 60 mm Hg for 3 hours) and severe metabolic acidosis (potassium 2.28 mmol/l, phosphate 0.21 mmol/l, pH 7.03, base excess -20.8 mmol/l). He was treated with massive fluid replacement (6.2 l in the first 12 hours), electrolyte substitution to counteract the marked hypokalaemia and hypophosphataemia, repeated defibrillation and antiarrhythmic drugs (lidocaine 240 mg/h and metoprolol twice 5 mg), as well as anticonvulsive treatment (diazepam, 10 mg twice, followed by midazolam 5 mg/h). Detoxication measures consisted initially of gastric lavage followed by high-dosage enteric administration of charcoal (210 g over 36 h), as well as haemoperfusion for 4 h. Full recovery was achieved and the patient was discharged in good health after 3 weeks.

[The initial results of lumbar dual-energy x-ray absorptiometry (DEXA) compared to double-energy quantitative computed tomography (DE-QCT)]. / Erste Ergebnisse der lumbalen Zweienergie-Röntgenabsorptiometrie (DEXA) im Vergleich mit der Doppelenergie-quantitativen Computertomographie (DE-QCT).

Bone density measurements were performed on 51 patients with suspected osteoporosis. We used DE-QCT and DEXA. The methods were compared with regard to deviation from normal. In 29 of the 51 patients there was more than half standard deviation and in 12 of the 51 cases there was more than a full standard deviation.

Comparative study on the thermostability of collagen I of skin and bone: influence of posttranslational hydroxylation of prolyl and lysyl residues.

Pepsin-solubilized collagen I from skin and bone was analyzed with regard to its thermal stability as a triple helical molecule in solution and after in vitro fibril formation. Collagen I from human control bone was compared with samples showing deficiencies or surplus in the degree of hydroxylation of lysine. The helix to coil transitions were studied by circular-dichroism measurements and limited trypsin digestion. Melting of fibrils from standardized in vitro self-assembly was investigated turbidimetrically. Human control bone collagen I has a maximum transition rate (Tm) at 43.3 degrees C in 0.05% acetic acid. This is 1.9 degrees C above control skin (Tm = 41.4 degrees C), most likely, due to a higher degree of prolyl hydroxylation--0.48 in bone vs. 0.41 in skin collagen I. Lysyl overhydroxylation of human and mouse bone collagen I appears to reduce the Tm slightly (approximately 1 degree C). Underhydroxylated bone collagen has a Tm which is 2 degrees C below control. Melting temperatures of in vitro formed fibrils are an indication for higher thermostability in parallel with an increase of lysyl hydroxylation. Accordingly, the melting temperature of such fibrils from human control skin, 49.3 degrees C, exceeds control bone by 1.4 degrees C. The degree of lysyl hydroxylation in these samples is 0.14 and 0.10, respectively. Further underhydroxylation (0.06) reduced it down to 45.4 degrees C, while extensive overhydroxylation did not continue to increase the thermal stability of fibrils.