Effect of riboflavin or pyridoxine deficiency on inflammatory response.

Inflammatory response has been assessed in riboflavin or pyridoxine deficient rats. Edema was increased by 54% in pyridoxine deficiency as compared to weight-matched control rats. Food restriction per se reduced the volume of edema by 63%. In pyridoxine deficiency, concentrations of thiobarbituric acid reactive substances (which indicate the extent of lipid peroxidation) increase by 30 and 43% respectively in the edematous tissues of the paw as well as in the wounded skin. Both these parameters were not affected by riboflavin deficiency. Activities of NADPH oxidase and superoxide dismutase in elicited leukocytes from peritoneal cavity were reduced by 54 and 52%, respectively, in riboflavin deficiency but were unaltered in pyridoxine deficiency. Superoxide level and acid phosphatase activity were not influenced by either of the deficiencies, whereas hydrogen peroxide level was increased by 48% in riboflavin deficiency. Food restriction did not affect leukocyte enzymes or the levels of reduced oxygen species. The data suggest that inflammation is enhanced in pyridoxine deficiency but not in riboflavin deficiency.     

Impaired collagen maturity in vitamins B2 and B6 deficiency--probable molecular basis of skin lesions

Solubility of collagen was increased and the proportion of insoluble collagen was reduced in the skin of both riboflavin as well as pyridoxine-deficient rats. Collagen content of the skin, and aldehyde concentration of salt-soluble collagen were also lower in the deficient groups. The alpha:beta subunit ratio of salt-soluble collagen was higher in riboflavin deficiency. In food-restricted weight-matched control groups, similar changes in collagen solubility, but of lesser magnitude were observed. Both food restriction and riboflavin deficiency decreased plasma PLP concentration. Increase in the solubility of collagen, decrease in the aldehyde content of soluble collagen and increase in the alpha:beta subunit ratio of soluble collagen, suggest that the maturation of collagen may be affected in pyridoxine or riboflavin deficiency. These molecular events may be etiologically related to the pathogenesis of the skin lesions in vitamin B2 or B6 deficiency.     

Ischemic heart disease as deficiency disease.

Four classes of agents capable of producing human illness have been identified: toxicity, heredity, infection and deficiency. The leading paradigm for the etiology and pathophysiology of ischemic heart disease in the 20th century was that of intoxication by too much of the wrong kind of dietary fat. This overemphasis on lipid metabolism persists because important data are neglected and because of inattention to details. For example, heart disease risk does not correlate with fat intake within nations in contrast to between nations. Also development of ischemic heart disease involves inter alia arterial spasm, cardiac rhythm, metabolism of connective tissue, glucose and homocysteine, plus paraoxonase activity and thrombus formation which generally are unaffected by dietary fat. Homocysteine thiolactone accumulates when homocysteine is high. This lactone specifically inhibits lysyl oxidase which depends on copper to catalyze cross linking of collagen and elastin in arteries and bone. The lactone is hydrolyzed by paraoxonase, activity of which can be decreased by copper deficiency. Just as cholesterol was an important focus for heart disease as intoxication, homocysteine can become an excellent focus for a paradigm shift to heart disease as deficiency because supplementation with several nutrients can alter homocysteine metabolism and decrease its plasma concentration. These supplements include betaine, copper, folate, pyridoxine and vitamin B-12. Opportunities for research on ischemic heart disease as deficiency disease are plentiful.     

High-performance liquid chromatographic quantification of allysine as bis-p-cresol derivative in elastin

Summary. The first step in normal cross-linking in elastin is the formation of α-aminoadipic-δ-semialdehyde, allysine, through oxidative deamination of specific peptidyl lysine by the enzyme lysyl oxidase (EC 1.4.3.13). For the analysis of allysine, allysine was derivatized with p-cresol. The derivatization was carried out by acid hydrolysis (6N HCl containing 5% (w/v) p-cresol at 110°C for 48 h) accompanied with the hydrolysis of elastin. A bis-p-cresol derivative of allysine was isolated from bovine ligamentum nuchae elastin hydrolysates, and was characterized by UV, FAB-MS and NMR. This derivative was identified as 2-amino-6,6-bis(2-hydroxy-5-methylphenyl)hexanoic acid. A rapid, sensitive reverse-phase high-performance liquid chromatographic method with UV detection was developed for the quantitative determination of allysine as its bis-p-cresol derivative. The lower limit of detection of the bis-p-cresol derivative was 58 pmol in the standard sample with a 20-μl injection at a signal-to-noise ratio of 3. This method was applied to the determination of allysine in bovine ligamentum nuchae, aorta, lung, and rat aorta elastin. The allysine content in rat aorta elastin dramatically increased from 1 week to 2 weeks of age. Received June 30, 2000 Accepted September 22, 2000     

Lysyl oxidase: a pituitary hormone-dependent enzyme.

Aldehyde-deficient non-crosslinked collagen obtained from lathyritic rats and collagen from penicillamine-treated rats, which is not deficient in aldehydes but the crosslinking of which is also inhibited, were implanted into the peritoneal cavity of hypophysectomized rats using the diffusion chamber technique. The enzyme lysyl oxidase which catalyses the aldehyde formation in certain lysyl residues of collagen and elastin was extracted from the skin of hypophysectomized rats. The activity of the enzyme was determined following its incubation with an L-[4,5-3H] lysine-labeled elastin substrate prepared from aortas of 17-day-old chick embryos. The result showed that the aldehyde deficient collagen did not crosslink while in the hypophysectomized animal indicating the lack of active lysyl oxidase in the rats. The enzyme activity in the skin of hypophysectomized animals was markedly reduced as compared with the controls indicating directly the dependance of lysyl oxidase activity on pituitary gland hormones.     

Changes in collagen cross-linking and lysyl oxidase by estrogen.

Dermal collagen solubility and lysyl oxidase activity of bones were measured in DDD mice of advancing age. Insoluble fractions of the dermal collagen increased more rapidly in females than in males after 5 weeks of age. Activity of the lysyl oxidase extracted from bones was higher in females than in males after 4 weeks of age. After sexual maturation, such sex differences were always observed in skin as well as in bone tissue. In other experimental animals, dermal collagen solubility was markedly decreased by estrogen treatment and lysyl oxidase was remarkably activated by estrogen in both skin and bone. Thus it is clear that estrogen stimulates the enzyme activity and accelerates the maturation of collagen and elastin in extracellular space.     

Decreased lysyl oxidase activity in the aneurysm-prone, mottled mouse.

Inbred mice bearing certain alleles at the Mottled locus have defects in connective tissue which result in weakness of skin and of blood vessels. Previous studies have established that cross-links in collagen and elastin are decreased in these animals due to impaired formation of lysine-derived aldehydes. Lysyl oxidase activity in extracts of skin is markedly lower in those prepared from affected animals than control mice. An inhibitor of lysyl oxidase is present in equal amounts in affected and control skins and does not account for diminished activity found in affected animals.     

Collagen cross-linking. Purification and substrate specificity of lysyl oxidase.

Lysyl oxidase is a specific amine oxidase that catalyzes the formation of aldehyde cross-link intermediates in collagen and elastin. In this study, lysyl oxidase from embryonic chick cartilage was purified to constant specific activity and a single protein band on sodium dodecyl sulfate acrylamide gel electrophoresis. This band had an apparent molecular weight of 62,000. The eluted protein cross-reacted with inhibiting antisera developed against highly purified lysyl oxidase. The highly purified enzyme was active with both insoluble elastin and embryonic chick skin or bone collagen precipitated as reconstituted, native fibrils. There was low activity with nonhydroxylated collagen, collagen monomers, or native fibrils isolated from lathyritic calvaria. The maximum number of aldehyde intermediates formed per molecule of collagen that became insoluble was two. These results indicate that lysyl oxidase has maximum activity on ordered aggregates of collagen molecules that may be overlapping associations of only a few collagen molecules across. Formation of aldehyde intermediates and cross-links during fibril formation may facilitate the biosynthesis of stable collagen fibrils and contribute to increased fibril tensile strength in vivo.     

Activities of liver microsomal fatty acid desaturases in zinc-deficient rats force-fed diets with a coconut oil/safflower oil mixture of linseed oil

The present study was conducted to investigate the effect of zinc deficiency on fatty acid desaturation in rats fed two different types of dietary fat, a mixture of coconut oil and safflower oil (7∶1, w/w, “coconut oil diet”) or linseed oil (“linseed oil diet”). In order to ensure an adequate food intake, all rats were force-fed by gastric tube. Zinc deficiency caused statistical significant reducion of Δ9-desaturase activity in liver microsomes of rats fed coconut oil diet and tendencial reduction (p<0.15) in rats fed linseed oil diet compared with control rats fed diets with the same type of fat. In agreement with this effect, zinc deficiency in the rats fed both types of dietary fat increased the ratio between total saturated and total monounsaturated fatty in liver phospholipids and liver microsomes. Zinc deficient rats on the coconut oil diet had unchanged Δ6-desaturase activity with linoleic acid as substrate and lowered activity with α-linolenic acid as substrate. In contrast, zinc deficient rats on the linseed oil diet had increased Δ6-desaturase activity with linoleic acid as substrate and unchanged activity with α-linolenic acid. Because linoleic acid is the main substrate for Δ6-desaturase in the rats fed coconut oil diet, and α-linolenic acid is the main substrate in the rats fed linseed oil diet, it is concluded that in vivo Δ6-desaturation was not changed by zinc deficiency in the rats fed both types of dietary fat. Activity of Δ5-desaturase was also not changed by zinc deficiency in the rats fed both dietary fats. Levels of fatty acids in liver phospholipids and microsomes derived by Δ4-, Δ5-, and Δ6-desaturation were not consistently changed by zinc deficiency in the rats fed both types of dietary fat. Thus, the enzyme studies and also fatty acid composition data of liver phospholipids and microsomes indicate that zinc deficiency does not considerably disturb desaturation of linoleic and α-linolenic acid. Therefore, it is suggested that similarities between deficiencies of zinc and essential fatty acids described in literature are not due to disturbed desaturation of linoleic acid in zinc deficiency. The present study also indicates that zinc deficiency enhances incorporation of eicosapentaenoic acid into phosphatidylcholine of rats fed diets with large amounts ofn-3 polyunsaturated fatty acids.     

The Effect of Diabetes On Lysyl Oxidase Activity and Extractability of Newly Synthesized Collagen in Rat Gingiva and Skin1

Lysyl oxidase activity and the extractability of recently synthesized collagen were measured in gingiva and skin, two tissues which exhibit altered pathophysiologic responses during diabetes. Both tissues showed a reduced content and extractability of newly synthesized, ¹⁴C-Hydroxyproline labeled, collagen after induction of streptozotocin-induced diabetes. Diabetes increased lysyl oxidase activity in skin and gingiva but the effect on gingiva was much less dramatic. The altered characteristics of interstitial collagen in the diabetic rat, including aging-like changes, appear to reflect multiple alterations in collagen metabolism.     

Effect of vitamin B-6 (pyridoxine) deficiency on lung elastin cross-linking in perinatal and weanling rat pups.

Weanling and perinatal rats were rendered vitamin B-6 (pyridoxine)-deficient. The rat pups were nursed from vitamin B-6-deficient or -sufficient dams and were killed at day 15 after parturition. The weanling rats were fed vitamin B-6-deficient or -sufficient diets and were killed after 5 weeks of treatment. Lung elastin from the groups of rats was then studied with respect to its content of lysine-derived cross-linking amino acids. Lung lysyl oxidase activity was also measured. B-6 deficiency decreased the number of lysine residues in elastin that were converted into the cross-linking amino acid precursor allysine. However, a more significant defect in cross-link formation was an apparent block in the condensation steps leading to the formation of desmosine. Desmosine was decreased, with an increase in the amounts of aldol condensation products (aldol CP) in elastin. It is proposed that the elevation in aldol CP results from the formation of thiazines, which are produced from the reaction between aldehyde and homocysteine. The concentration of homocysteine is significantly elevated in vitamin B-6-deficient rats.     

Inflammation-associated lysyl oxidase protein expression in vivo, and modulation by FGF-2 plus IGF-1

 Lysyl oxidase is the extracellular enzyme that catalyzes oxidative deamination of peptidyl-lysine residues in elastin precursors, and lysine and hydroxylysine residues in collagen precursors to form peptidyl-aldehydes. These aldehydes then spontaneously condense to crosslink collagen and elastin and thereby allow the formation of a mature and functional extracellular matrix. In the present study, cryosections made from aseptic immune-induced periapical lesions experimentally generated in laboratory rats were examined by immunohistochemistry to investigate whether lysyl oxidase protein expression is altered in inflamed oral tissues. Periapical lesions are experimentally induced endodontic lesions of tooth roots. In addition, the effect of administration of a mixture of fibroblast growth factor (FGF)-2 and insulin-like growth factor (IGF)-1 into these lesions on lysyl oxidase expression was determined. Lysyl oxidase expression was found to be increased in non-mineralized connective tissue adjacent to inflamed lesions. Morphometric analyses indicated that maximum lysyl oxidase expression occurred at a discrete distance from the lesion not exceeding 350 μm from the inflammatory cells. Staining was associated with mesenchymal cells with a fibroblastic morphology. No lysyl oxidase staining was found near teeth where no lesion was induced. Application of a mixture of FGF-2 and IGF-1 resulted in a further twofold increase in lysyl oxidase expression. These results provide a new in vivo model to study lysyl oxidase regulation, and suggest that inflammatory cells may control lysyl oxidase expression in oral tissues, possibly by a mechanism involving secretion of cytokines and other factors, probably contributing to the regulation of extracellular matrix accumulation. Accepted: 19 December 1998     

Glutathione reductase activity and pyridoxine (pyridoxamine) phosphate oxidase activity in the red cell

The red-cell enzymes, glutathione reductase (FAD-dependent) and pyridoxine (pyridoxamine) phosphate oxidase (FMN-dependent), were studied in control subjects. The wide range the glutathione reductase activity correlated inversely with the percentage stimulation by FAD added in vitro, and with pyridoxine (pyridoxamine) phosphate oxidase activity. Both enzymes were stimulated after ingestion of riboflavin. The results support the suggestion that the rate of metabolism of riboflavin in the red cell controls the activity of both enzymes, and the rate of red-cell metabolism of vitamin B-6.     

Pyridoxine supplementation corrects vitamin B6 deficiency but does not improve inflammation in patients with rheumatoid arthritis

Patients with rheumatoid arthritis have subnormal vitamin B6 status, both quantitatively and functionally. Abnormal vitamin B6 status in rheumatoid arthritis has been associated with spontaneous tumor necrosis factor (TNF)-α production and markers of inflammation, including C-reactive protein and erythrocyte sedimentation rate. Impaired vitamin B6 status could be a result of inflammation, and these patients may have higher demand for vitamin B6. The aim of this study was to determine if daily supplementation with 50 mg of pyridoxine for 30 days can correct the static and/or the functional abnormalities of vitamin B6 status seen in patients with rheumatoid arthritis, and further investigate if pyridoxine supplementation has any effects on the pro-inflammatory cytokine TNF-α or IL-6 production of arthritis. This was a double-blinded, placebo-controlled study involving patients with rheumatoid arthritis with plasma pyridoxal 5'-phosphate below the 25th percentile of the Framingham Heart Cohort Study. Vitamin B6 status was assessed via plasma and erythrocyte pyridoxal 5'-phosphate concentrations, the erythrocyte aspartate aminotransferase activity coefficient (αEAST), net homocysteine increase in response to a methionine load test (ΔtHcy), and 24 h urinary xanthurenic acid (XA) excretion in response to a tryptophan load test. Urinary 4-pyridoxic acid (4-PA) was measured to examine the impact of pyridoxine treatment on vitamin B6 excretion in these patients. Pro-inflammatory cytokine (TNF-α and IL-6) production, C-reactive protein levels and the erythrocyte sedimentation rate before and after supplementation were also examined. Pyridoxine supplementation significantly improved plasma and erythrocyte pyridoxal 5'-phosphate concentrations, erythrocyte αEAST, urinary 4-PA, and XA excretion. These improvements were apparent regardless of baseline B6 levels. Pyridoxine supplementation also showed a trend (p < 0.09) towards a reduction in post-methionine load ΔtHcy. Supplementation did not affect pro-inflammatory cytokine production. Although pyridoxine supplementation did not suppress pro-inflammatory cytokine production in patients with rheumatoid arthritis, the suboptimal vitamin B6 status seen in rheumatoid arthritis can be corrected by 50 mg pyridoxine supplementation for 30 days. Data from the present study suggest that patients with rheumatoid arthritis may have higher requirements for vitamin B6 than those in a normal healthy population.     

Inhibition of lysyl oxidase and prolyl hydroxylase activity in glucocorticoid treated rats

Triamcinolone diacetate produced a dose dependent decrease in lysyl oxidase activity in the skin of new born rats when administered over a three day period. Maximum inhibition by this glucorticoid resulted in less than 10% of control lysyl oxidase activity. A similar though less dramatic effect was observed on skin prolyl hydroxylase activity. These results suggest that the antianabolic effect of glucocorticoids on collagen synthesis extend to enzymes involved in the intra- and extracellular modifications of collagen.     

High-Yielding Enzymatic α-Glucosylation of Pyridoxine by Marine α-Glucosidase from Aplysia fasciata

We recently succeeded in the identification and purification of an interesting marine exo-α-glucosidase (EC 3.2.1.20) from the anaspidean mollusc Aplysia fasciata. The enzyme was characterized by good transglycosylation activity toward different acceptors using maltose as donor. High-yielding enzymatic α-glycosylation of pyridoxine using this marine enzyme is reported here; the reaction has been optimized, reaching 80% molar yield of products (pyridoxine monoglucosides 24 g/l; pyridoxine isomaltoside 35 g/l). High selectivity toward the 5′ position is observed for both monoglucoside and disaccharide formation. This is the first report describing the enzymatic production of pyridoxine isomaltoside.     

The human lysyl oxidase-like 2 protein functions as an amine oxidase toward collagen and elastin

The lysyl oxidase-like 2 (LOXL2) protein is a human paralogue of lysyl oxidase (LOX) that functions as an amine oxidase for formation of lysine-derived cross-links found in collagen and elastin. In addition to the C-terminal domains characteristic to the LOX family members, LOXL2 contains four scavenger receptor cysteine-rich (SRCR) domains in the N-terminus. In order to assess the amine oxidase activity of LOXL2, we expressed a series of recombinant LOXL2 proteins with deletions in the SRCR domains, using an Escherichia coli expression system. All of the purified recombinant LOXL2 proteins, with or without the SRCR domains in the N-terminus, showed significant amine oxidase activity toward several different types of collagen and elastin in in vitro amine oxidase assays, indicating deletion of the SRCR domains does not interfere with amine oxidase activity of LOXL2. Further, amine oxidase activity of LOXL2 was not susceptible to inhibition by β-aminopropionitrile, an irreversible inhibitor of LOX, suggesting a different enzymatic mechanism between these two paralogues.     

A recombinant bait region mutant of human α2-macroglobulin exhibiting an altered proteinase-inhibiting spectrum

Alpha 2-macroglobulin (α2M), a plasma glycoprotein produced in the liver, inhibits a variety of proteinases and thus considered to play important homeostatic roles in the body. This broad inhibitory spectrum has been explained by the trapping theory by which a proteinase recognizes a region of 25–30 amino acid peptide in α2M called bait region and cleaves it, leading to the conformational change of α2M, and to the subsequent entrapment and inhibition of the proteinase. We constructed α2M cDNAs with mutated DNA sequences in the bait region, and obtained recombinant CHO cell lines producing either wild type α2M, or mutant α2Ms, i.e., α2M/K692 and α2M/K696, each with substitution of Arg with Lys at codons 692 and 696, respectively. We tested if lysyl endopeptidase is not inhibited by wild type α2M, but could be inhibited by these engineered mutant α2Ms. Thus, recombinant α2M/K696 protein successfully inhibited lysyl endopeptidase activity, while recombinant α2M/K692 protein was not sensitive to lysyl endopeptidase, suggesting that not all bait region peptide bonds can equally be accessible and susceptible to proteinases. The present results not only provided the trapping theory with additional supportive evidence, but the first experimental evidence for the value of engineered α2M-derived proteinase inhibitor with an artificial proteinase inhibitory spectrum of potential industrial and/or therapeutic usefulness. This revised version was published online in August 2006 with corrections to the Cover Date.     

Staphylococcus aureus in chronic and recurrent infections

Ninety-fourStaphylococcus aureus strains isolated from chronic and recurrent skin and respiratory tract infections were investigated for several virulence factor expressions. Production of protein A was noticed in all of the tested strains in amounts from less than 0.1 to more than 2.5 ng per 10⁶ bacterial cells. The percentage of the extracellularly produced protein A was found to lie between 4.5 and 27.8%. Two strains (both from the respiratory tract) produced more than 50 % of protein A in the extracellular form and one strain did not produce any detectable amount of the extracellular protein A; 99 % of the tested strains produced the clumping factor, 96% staphylocoagulase, 79 % staphylokinase and 90 % gelatinolytic activity; 79 % produced α-toxin exclusively or in combination with δ- or β-toxin; 8 % of strains produced β-toxin. There were differences in β-toxin production between strains from the respiratory tract (5 %) and skin infections (25 %). δ-Toxin was produced by 53 % of the strains. In each of the tested strains a complex of virulence factors was detected. The importance of inactivated extracellular products (especially α- and δ-toxin and in the case of skin infections also β-toxin) as components of staphylococcal whole-cell vaccine was suggested. Dedicated to Professor C. John on the occasion of his 75th birthday     

The inhibition of lysyl oxidase in vivo by isoniazid and its reversal by pyridoxal. Effect on collagen cross-linking in the chick embryo.

Isonicotinic acid hydrazide (isoniazid) causes a large increase in the salt-solubility of collagen when injected into chick embryos; this change is accompanied by the inactivation of lysyl oxidase (EC 1.4.3.13), the enzyme responsible for initiating cross-link formation in collagen and elastin. In addition, isoniazid markedly decreases the liver content of pyridoxal phosphate. The depletion of pyridoxal phosphate takes approx. 6 h, whereas the inhibition of lysyl oxidase and the increase in collagen solubility occur more slowly. A reversal of these effects of isoniazid can be produced by the subsequent injection of a stoichiometric amount of pyridoxal, supporting the role of pyridoxal as a cofactor for lysyl oxidase. Treatment of chick embryos with beta-aminopropionitrile, an irreversible inhibitor of lysyl oxidase, causes an inhibition of the enzyme, which begins to recover within 24 h but which is not affected by the administration of pyridoxal; with isoniazid inhibition, however, lysyl oxidase activity does not show any sign of recovery by 48 h. It is proposed that isoniazid may cause the inhibition of lysyl oxidase by competing for its obligatory cofactor, pyridoxal phosphate. The potential clinical implications in the therapeutic control of fibrosis are briefly discussed.