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Alpacas (Vicugna pacos) produce a high-value fibre and leather that is increasingly sought after for the production of luxury goods. Although several studies have addressed the genetics and gene expression of alpaca skin, particularly those associated with colour, little is known about the structural and molecular compositions of alpaca skin. In this study, alpaca skin was analysed using small angle X-ray scattering (SAXS), analytical and physical methods. Using SAXS, the axial periodicity (65.1 nm) and intermolecular lateral packing distance (1.47 nm) of collagen in alpaca skin are determined to be similar to the previous studies on skins from other animal species. The collagen crosslinking profile in alpaca skin shows the presence of six crosslinks including pyridinoline and deoxypyridinoline and this is different from other animal skins. We hypothesised that the biosynthesis of pyridinoline and deoxypyridinoline in biological tissues is driven by a combination of several factors including amino acid sequence of collagen type I and lysyl hydroxylase activity. In this work, we showed that alpaca skin collagen crosslinks are diverse and their relative concentrations are different from other animal skins. We also detected histidinohydroxylysinonorleucine crosslink in alpaca skin which lacks histidine in its C-terminal telopeptide of type I collagen.

Lysates of human skin fibroblasts harvested without the use of trypsin do not contain detectable proteolytic activity, but when trypsin is used, lysates may contain activity equal to 10 ng of trypsin/10 7 cells. The amount of cell lysate ordinarily examined for collagen prolyl and lysyl hydroxylase activity is sufficiently small that such amounts of trypsin have no observable effect on the unhydroxylated collagen substrate. Larger amounts of trypsin cause proteolysis of the unhydroxylated collagen substrate and a reduction of both prolyl and lysyl hydroxylation with lysyl hydroxylation more affected at low trypsin concentration than prolyl hydroxylation.

Studies with confluent human skin fibroblasts maintained in 0.5% serum supplemented medium have given new insight into the regulatory influences of ascorbate. These include a reduction of prolyl hydroxylase activity, a stimulation of lysyl hydroxylase activity, and an acceleration of collagen production. The lack of parallel between prolyl hydroxylase activity and collagen production indicates that the rate of collagen synthesis is not controlled by the level of prolyl hydroxylase.

The influence of diabetes on gingival inflammation was studied through its effect on prolyl and lysyl-hydroxylase activities and on tissue-collagen content. Inflammation induced for 7 days with either endotoxin or antigen-antibody complexes reduced the activity of both enzymes by about 45 per cent, and decreased the concentration of soluble and insoluble collagens. Diabetes alone decreased the enzyme activity by more than 50 per cent but prevented the loss of the soluble and insoluble collagens which occurs during inflammation. The complex interaction of diabetes and inflammation on collagen metabolism in gingival tissue may be explained in part by abnormalities of collagen synthesis; alterations in collagen maturation and degradation, and in leukocyte function, may also occur.

In the absence of ascorbic acid, confluent human skin fibroblasts incubated in 0.5% serum-supplemented medium had one-third of the level of lysyl hydroxylase activity of cells incubated in media containing high serum concentrations (5-20%). This difference appeared to be due to a decline in the enzyme activity following serum deficiency, and was largely abolished by addition of ascorbic acid to the medium. The effect of serum deficiency was slow, manifesting in 48 h at the earliest, and was completely reversed by replenishing the medium with serum. Prolyl hydroxylase activity was independent of serum concentration, both in the absence and in the presence of ascorbic acid in the culture medium.

Two important findings related to the effect of minoxidil on human skin fibroblasts in culture are reviewed. Treatment of cells with minoxidil is associated with a specific loss of lysyl hydroxylase activity; this loss occurs gradually and is reversed by removing minoxidil from the culture medium. Experiments with inhibitors of protein and RNA synthesis reveal that minoxidil may inhibit the synthesis of lysyl hydroxylase by acting at the transcriptional level. Treatment of cells with minoxidil is also associated with inhibition of proliferation, without any sign of cytotoxicity. This effect of minoxidil is accompanied by inhibition of DNA synthesis. Since collagen is the major product of fibroblast activity and lysyl hydroxylase catalyzes a crucial reaction in collagen biosynthesis, the combined effects of minoxidil offer the potential for its use as an antifibrotic agent. Thus, minoxidil may prove to be beneficial in treating skin conditions associated with collagen accumulation.

The uptake of ascorbic acid by cultured skin fibroblasts from two siblings with Ehlers-Danlos syndrome type VI, in extracts of which there is no deficit in lysyl hydroxylase activity, was found to be normal. Decreased availability of ascorbic acid to lysyl hydroxylase in cellula cannot, therefore, provide an explanation for the disorder in these patients, the cause of which remains obscure.

In confluent human skin fibroblasts maintained in 0.5% serum-supplemented medium. L-ascorbate specifically stimulated the rate of incorporation of labeled proline into total collagenase-sensitive protein, without changing the specific activity of the intracellular free proline. This influence of ascorbate reached a maximum at 30 microM and continued for at least 4 days, resulting in a 4-fold increase. The ascorbate effect occurred in cells at both confluent and subconfluent densities and was evident at all serum concentrations from 0.5-20%. The effect was independent of duration of the radioactive pulse between 2-6 h. D-Ascorbate, D-isoascorbate, and L-dehydroascorbate also stimulated collagen synthesis but at considerably higher concentrations, i.e., 250-300 microM. The stimulation of collagen synthesis by ascorbate and its analogs was accompanied by a decline in prolyl hydroxylase activity and a rise in lysyl hydroxylase activity; again L-ascorbate was found to be most effective. Dimethyltetrahydropterine and L-lactate failed to produce these effects.

Five disorders of collagen have been characterized. In four of these disorders the patients have had symptoms of Ehlers Danlos. Three families with lysyl hydroxylase deficiency have previously been reported. We are reporting an additional case diagnosed in infancy. This infant was investigated as a floppy baby but nerve and muscle were normal. Lysyl hydroxylase activity measured in sonicates of dermal fibroblasts was 10% of normal. The deficiency of hydroxylysine results in abnormal cross-linking of collagen molecules which may account for the symptomatology. The patient we are reporting eventually developed symptoms of Ehlers Danlos. This type of Ehlers Danlos is an autosomal recessive disorder in which one might expect to make an intrauterine diagnosis. Collagen abnormalities may thus be a cause of floppy baby syndrome and the delineation of a specific defect should be the goal of diagnostic studies.