R. Milbradt

Antioxidant Inhibition of Skin Inflammation Induced by Reactive Oxidants: Evaluation of the Redox Couple Dihydrolipoate/Lipoate

Reactive oxygen species play an important role in mediating skin inflammation, and antioxidants may provide protection. We investigated the anti-inflammatory activity of natural antioxidants, such as superoxide dismutase, catalase, trolox (a water-soluble tocopherol analog) and the redox couple dihydrolipoate/lipoate in skin. Furthermore we compared the anti-inflammatory potency of natural R and racemic dihydrolipoate, as well as R and S lipoate. Skin inflammation in hairless mice was induced by intradermal injection of the hydrogen peroxide producing enzyme glucose oxidase (GOD) or by topical application of the prooxidant drug anthralin. Intradermal injection of the antioxidants inhibited skin inflammation caused by GOD (catalase, dihydrolipoate) and anthralin (trolox, superoxide dismutase, dihydrolipoate). There was no statistically significant difference between the anti-inflammatory activity of the natural R and racemic dihydrolipoate. R or S lipoate did not inhibit skin inflammation when injected intradermally. In feeding experiments, however, R lipoate significantly inhibited GOD-mediated skin inflammation, while S lipoate was only marginally protective. We conclude that (1) several natural antioxidants such as catalase, superoxide dismutase and dihydrolipoate have anti-inflammatory properties in dermatitis induced by reactive oxidants, (2) lipoate (oxidized dihydrolipoate) has skin anti-inflammatory activity when administered orally and (3) naturally occurring R lipoate is a more potent anti-inflammatory agent than the non-physiological S lipoate.

[30] Sensitive high-performance liquid chromatography techniques for simultaneous determination of tocopherols, tocotrienols, ubiquinols, and ubiquinones in biological samples

Publisher Summary Lipophilic antioxidants are responsible for the protection of cell membranes against lipid peroxidation. Vitamin E is the major lipophilic antioxidant in plasma, membranes, and tissues and is the collective name for the eight naturally occurring molecules (four tocopherols and four tocotrienols) that exhibit α-tocopherol activity. This chapter discusses the sensitive high-performance liquid chromatography techniques for simultaneous determination of tocopherols, tocotrienols, ubiquinols, and ubiquinones in biological samples. The method described in this chapter allows the extraction and detection of the various vitamin E forms along with ubiquinol/ubiquinone in a single aliquot of tissue. The unexpected distribution of vitamin E forms in different tissues exemplifies that the selective determination of the major lipophilic antioxidants is important for understanding the relationship among diet, oxidative stress, and cellular regulatory processes.

Chemiluminescence in semen of infertile men

Summary. Luminol‐dependent chemiluminescence (CL) can be used to determine the production of reactive oxygen species (ROS) by cells. Enhanced formation of ROS in human semen was reported to be of pathological significance for a disturbed sperm function. To investigate incidence of elevated CL‐signals in semen samples and their correlation to conventional semen parameters, CL‐signals in the semen of both 49 consecutive infertile men and 20 controls were measured. Semen was analysed according to WHO‐criteria including bovine mucus‐penetration‐ and water‐test. A CL‐signal of 1.5 × 105 counts min−1/2 × 106 spermatozoa was considered to be the upper normal limit. The CL in infertile mens semen was elevated with statistically significant differences in oligozoospermia patients/controls (P < 0.0001) and normozoo‐spermia patients/controls (P < 0.05). In the group with elevated CL‐signals, a higher percentage of spermatozoa with a pathologic morphology was detected (P = 0.05). In the groups with pathologic results of eosin‐ and water‐tests, the CL‐counts were elevated (P < 0.006; P < 0.03). The spermatozoa motility in the group with elevated CL‐counts was significantly reduced after 4 h (P < 0.05). The CL‐signals correlated inversely with the results of the bovine mucus‐penetration‐test (r = ‐0.67, P < 0.0001). In conclusion, semen samples of 28% of our patients showed elevated CL‐signals; these were associated with pathological results of membrane integrity‐tests. The negative correlation of CL with the results of Penetrak®‐test reflects its importance to depict the functional capacity of spermatozoa.

Nitroxide radical biostability in skin

Nitroxide radicals are important chemical tools in dermatologic research (e.g., for studying biophysical properties of skin lipids and epidermal membranes with the method of electron paramagnetic resonance, EPR, spectroscopy). However, nitroxides may loose their paramagnetic properties in biological tissues, which could limit their usefulness in biomedical applications. We analyzed the biostability of various chemical types of nitroxide radicals in keratinocytes, epidermis homogenate, and intact skin. EPR signal loss of imidazoline, pyrrolidine, piperidine, and oxazolidine nitroxides is attributed to their reduction to the corresponding hydroxylamine. The rate of nitroxide reduction in skin varies considerably with nitroxide ring structure and substitution. The order of nitroxide stability in isolated human keratinocytes, mouse epidermis homogenate, and intact mouse and human skin is imidazoline > pyrrolidine > di-t-butylnitroxide (DTBN) > piperidine > oxazolidine. Cationic nitroxides are reduced much faster than neutral or anionic probes, presumably due to transmembrane electron shuttle or internalization. The results indicate that imidazoline- and pyrrolidine-type nitroxides should be used when high biostability of nitroxides is needed. Piperidine-type nitroxides are versatile probes for studying one-electron transfer reactions in skin.

Oxidative imbalance in HIV infected patients

We present an outline of the complex interplay of oxidants and antioxidants in infectious diseases in general, and in particular with reference to the HIV infection, and subsequent opportunistic infections. Viral and opportunistic infections may directly or indirectly cause an imbalance in prooxidant/antioxidant mechanisms and result in generation of increased steady state concentrations of reactive oxidants. In HIV patients a prooxidant state could lead to a self-perpetuation of infection via stimulated expression of genes carrying the virus genome, and subsequently to immunosuppression, and promotion of initiated cells to neoplastic growth.

Cutaneous mucinosis of infancy: is it a real entity or the paediatric form of lichen myxoedematosus (papular mucinosis)?

We describe a girl presenting with a childhood dermal mucinosis in which we had the unique opportunity to find all the transitional histological features of lichen myxoedematosus (papular mucinosis), from its early focal mucin deposition in the reticular dermis to its late findings of interstitial mucin deposition, dermal fibrosis and fibroblast proliferation. Her father reported having had similar lesions when he was a child, which completely disappeared during adolescence. This case, and a re‐evaluation of the literature, suggests that cases of cutaneous mucinosis of infancy that are not hamartomatous conditions such as mucinous naevi are in fact the infantile presentation of lichen myxoedematosus (papular mucinosis) and, in addition to other cases in the literature, suggests a genetic and familial factor in lichen myxoedematosus (papular mucinosis).

CLASTOGENIC FACTORS IN PLASMA OF HIV-1 INFECTED PATIENTS

The objective of this study was to investigate the clastogenic activity of plasma ultrafiltrates from HIV-1 infected patients. Clastogenic factors are chromosome-damaging agents with low molecular weight (< 10,000 daltons) which cause chromosome aberrations, sister chromatid exchanges, DNA strand breakage, and gene mutation. They have first been described in the plasma of irradiated persons, but they are also found in hereditary breakage syndromes and chronic inflammatory diseases with autoimmune reactions. Their formation and their clastogenic effects are modulated by superoxide anion radicals. We analyzed a total of 22 HIV-1 positive patients in comparison to 20 reference plasma samples from healthy HIV negative blood donors of similar age. The plasma ultrafiltrates (filter cutoff 10,000 daltons) from patients induced a statistically significant increase in chromosomal breakage in the cytogenetic test system (20.5 +/- 6.8 aberrations per 100 cells), while no increase was observed in test cultures exposed to plasma ultrafiltrates from healthy blood donors (6.3 +/- 2.9 aberrations per 100 cells). The breakage values were slightly, but not significantly, lower in the 10 patients with more than 200 T-helper cells/ml (18 +/- 4 aberrations per 100 cells), than in the 12 patients with less than 200 T-helper cells/ml (22.3 +/- 7.9 aberrations per 100 cells). HIV patients with high clastogenic activity (induction of more than 20 aberrations per 100 cells, range 20 to 39) showed higher plasma levels for malondialdehyde than those with lower clastogenic activity (less than 20 aberrations per 100 cells, range 12 to 18). However, the difference was statistically not significant. Another lipid peroxidation product, 4-hydroxynonenal, was increased equally in both groups. There were no significant differences in water- and lipid-soluble plasma antioxidants between the low- and high-breakage group. In agreement with previous findings, the clastogenic effects of plasma ultrafiltrates in the test cultures were reduced by the antioxidant enzyme superoxide dismutase. The presence of clastogenic factors in the plasma of HIV patients is further evidence for a prooxidant state in these persons. Since clastogenic factor formation appears to occur at an early stage of the disease, it may be significant for virus release or activation, because of the superoxide anion stimulating effects of clastogenic factors. From a practical standpoint, clastogenic factors may be useful for evaluation of promising drugs.

On the interaction between anthralin and mitochondria: a revision

SummaryAnthralin is an inhibitor of oxidative phosphorylation at concentrations found in vivo. ADP-stimulated oxygen consumption is diminished. Consequently, the rate of ATP synthesis is reduced and mitochondrial ATP content declines. Neither the isolated ATPase (F1Fo-ATPase), nor the mitochondrial membrane-bound ATPase are influenced by the drug. Respiration under resting conditions is not affected. The experimental data unequivocally indicate that anthralin is not an uncoupler of oxidative phosphorylation, as previously stated. Furthermore, the interpretation that respiratory deficiency induced in yeast strains by anthralin is a consequence of petite mutations has to be reconsidered. Under in vivo conditions, anthralin inhibits respiration per se. Our experiments, including the electron spin resonance spectroscopy, reveal that anthralin alters mitochondrial membrane structure and function simultaneously. A redox or free-radical mediated step may be involved. In consequence, inhibition of ATP production occurs which may become the limiting factor for increased cellular metabolism in psoriasis.

Multifunctional analysis of the interaction of anthralin and its metabolites anthraquinone and anthralin dimer with the inner mitochondrial membrane.

SummaryWe studied the interaction of the antipsoriatic compound anthralin (1.8-dihydroxy-9-anthrone), and its metabolites anthraquinone (1.8-dihydroxy-9.10-anthraquinone) and anthralin dimer (1.8.1′.8′.-tetrahydroxy-10.10′-bis-9[10]-dianthrone) with the inner mitochondrial membrane. Mitochondrial membrane functions such as ubiquinone redox equilibria, redox status of iron sulfur clusters, cyanide-sensitive and cyanide-insensitive oxygen consumption, adenosine triphosphate (ATP) synthesis, ATP hydrolysis, and adenine nucleotide content of mitochondria were analyzed. Anthralin is an inhibitor of mitochondrial oxygen uptake in the presence of ADP and substrate (cyanide-sensitive respiration), inhibits ATP synthesis without affecting ATP hydrolysis, and depletes mitochondria of ATP. Anthralin dimer is a much weaker inhibitor of mitochondrial functions and anthraquinone is almost inactive. Anthralin, but not anthraquinone and anthralin dimer, reverses uncoupler stimulated oxygen consumption, stimulates cyanide-insensitive respiration, reduces mitochondrial ubiquinone-9 and -10 to the corresponding ubiquinols and reduces mitochondrial iron sulfur clusters. Anthralin may induce formation of reactive oxygen species by enhancing autoxidation of mitochondrial components and/or by catalyzed oxidation of anthralin. Taken together, anthralin acts as an electron donor to inner mitochondrial membrane associated redox components, inhibits the electron transport chain, and has an oligomycin-like effect. Anthralin dimer and anthraquinone do not function as electron donors and act by a different reaction mechanism. Respiratory measurements in human keratinocytes revealed similar results as obtained with isolated mitochondria. We suggest that modulation of membrane redox status may be a common concept of anthralin action in target cells such as keratinocytes and neutrophils.

Vitiligo treatment with phenylalanin/UV A. Follow-up studies after 5 years

Zusammenfassung. Seit 1983 wird zur Behandlung der Vitiligo Phenylalanin in Kombination mit UV-A-Bestrahlung (PAUVA) vermehrt eingesetzt. Langzeitergebnisse liegen bisher nicht vor. Um den Langzeitverlauf und die Langzeitnebenwirkungen zu erfassen, wurden 41 Patienten 5 Jahre nach PAUVA-Therapie angeschrieben. 25/41 konnten nachuntersucht werden. 11/25 (44%) zeigten einen anhaltenden Therapieerfolg. Eine Depigmentierung sowohl während als auch nach Therapieende trat bei 16/25 (64%) auf. 52% waren subjektiv zufrieden und würden die Therapie wiederholen, 68% würden diese weiterempfehlen. Als prognostisch günstige Parameter für ein gutes Ansprechen auf die Therapie konnten ein Befall der Körperoberfläche <25%, ein Vitiligobeginn vor dem 21. Lebensjahr, eine generalisierte stammbetonte Verteilung und UV-Bestrahlungen über längere Zeiträume angesehen werden. Langzeitnebenwirkungen wurden nicht festgestellt. Die PAUVA-Therapie kann demnach für bestimmte Patienten eine hilfreiche therapeutische Alternative darstellen.Summary. Since 1983 the administration of phenylalanine combined with UVA exposure (PAUVA) has been a well-known therapy for vitiligo. We have found no retrospective studies on this therapy. To document the long-term results and side effects, we performed a retrospective study on 41 patients who had received PAUVA therapy about 5 years ago. Examination was possible in 25 of the 41 patients, and 11 of them (44%) had permanent repigmentation. Depigmentation either during or after PAUVA therapy was recognized in 16 of the 25 patients (64%). In 52% of cases the patients were satisfied with the therapy and would repeat it; 68% would recommend it. Positive features in prognosis, i.e. indicative of good repigmentation, were vitiligo extending over less than 25% of the body surface, onset of vitiligo before the age of 21, generalized and symmetrical distribution and a long duration of UV therapy. None of our patients developed long-term side effects. PAUVA therapy is demonstrably a therapeutic alternative for certain patients.