Sleep and Circadian Rhythms of Temperature and Urinary Excretion on a 22.8 hr “Day”

Two groups of subjects (total N = 6) were studied in an isolation chamber for a period of 3 weeks whilst living on a 22.8 hr “day”. Regular samples of urine were taken when the subjects were awake, deep body temperature was recorded continuously and polygraphic EEG recordings were made of alternate sleeps. The excretion in the urine of potassium, sodium, phosphate, calcium and a metabolite of melatonin were estimated.

Measurements of the quantity and quality of sleep were made together with assessments of the temperature profiles associated with sleep. In addition, cosinor analysis of circadian rhythmicity in urinary variables and temperature was performed.

The 22.8 hr “days” affected variables and subjects differently. These differences were interpreted as indicating that the endogenous component of half the subjects adjusted to the 22.8 hr “days” but that, for the other three, adjustment did not occur. When the behaviour of different variables was considered then some (including urinary potassium and melatonin, sleep length and REM sleep) appeared to possess a larger endogenous component than others (for example, urinary sodium, phosphate and calcium), with rectal temperature behaving in an intermediate manner. In addition, a comparison between different rhythms in any subject enabled inferences to be drawn regarding any links (or lack of them) that might exist between the rhythms. In this respect also, there was a considerable range in the results and no links between any of the rhythms appeared to exist in the group of subjects as a whole.

Two further groups (total N=8) were treated similarly except that the chamber clock ran at the correct rate. In these subjects, circadian rhythms of urinary excretion and deep body temperature (sleep stages and urinary melatonin were not measured) gave no evidence for deterioration. We conclude, therefore, that the results on the 22.8 hr “day” were directly due to the abnormal “day” length rather than to a prolonged stay in the isolation chamber.     

Circadian and circannual rhythms of hormonal variables in elderly men and women

A group of fourteen men (73 ± 5 yr of age), and eighteen women (77 ± 7 yr of age) institutionalized at the Berceni Clinical Hospital, Bucharest, Romania, were studied over a 24-hr span once during each season (winter, spring, summer and fall). All subjects followed a diurnal activity pattern with rest at night and ate three meals per day with breakfast at about 0830, lunch at about 1300 and dinner at about 1830. The meals were similar, although not identical for all subjects during all seasons. On each day of sampling blood was collected at 4-hr intervals over a 24-hr span. Seventeen hormonal variables were determined by radioimmunoassay. Statistically significant circadian rhythms were detected and quantitated by population mean cosinor analysis in pooled data from all four seasons in both sexes for ACTH, aldosterone, Cortisol, C-peptide, dehydroepiandrosterone-sulfate (DHEA-S), immunoreactive insulin, prolactin, 17-OH progesterone, testosterone, total T₄ and TSH. In women, estradiol and progesterone also were determined and showed a circadian rhythm during all seasons. Total T, and FSH showed circadian rhythm detection by cosinor analysis in the men only; LH showed no consistent circadian rhythm as group phenomenon in men or women.

A circannual rhythm was detected using the circadian means of each subject at each season as input for the population mean cosinor in the women for ACTH, C-peptide, DHEA-S, FSH, LH, progesterone, 17-OH progesterone and TSH. In the men, a circannual rhythm was detected for ACTH, FSH, insulin, LH, testosterone and T₃. There were phase differences between men and women in ACTH, FSH and LH. In those functions in which both the circadian and circannual rhythms were statistically significant, a comparison of the amplitudes showed in the women a higher circannual rather than circadian amplitude for DHEA-S. In 17-OH progesterone, TSH and C-peptide, the circadian amplitude in women was larger. In men, the circannual amplitude of T₃ was larger than the circadian amplitude and in insulin the circadian amplitude was larger than the circannual amplitude. There was no statistically significant difference between the circadian and circannual amplitudes in the women in ACTH and progesterone and in the men in ACTH and testosterone.     

Plasma Corticosterone, Motor Activity and Metabolic Circadian Patterns in Streptozotocin-Induced Diabetic Rats

Experiments were conducted in male rats to study the effects of streptozotocin-induced diabetes on circadian rhythms of (a) plasma corticosterone concentrations; (b) motor activity; and (c) metabolic patterns. Animals were entrained to LD cycles of 12: 12 hr and fed ad libitum.

A daily rhythm of plasma corticosterone concentrations was found in controls animals with peak levels at 2400 hr and low values during the remaining hours. This rhythm was statistically confirmed by the cosinor method and had an amplitude of 3.37μg/100 ml and the acrophase at 100 hr. A loss of the normal circadian variation was observed in diabetic animals, with a nadir at the onset of light period and high values throughout the remaining hours; cosinor analysis of these data showed no circadian rhythm, delete and a higher mean level than controls.

As expected, normal rats presented most of their motor activity during the dark period with 80+ of total daily activity; the cosinor method demonstrated a circadian rhythm with an amplitude of 60+ of the mean level and the acrophase at 0852 hr. Both diabetic and control rats showed a similar activity during the light phase, but diabetic animals had less activity than controls during the night and their percentage of total daily activity was similar in both phases of the LD cycle (50+ for each one). With the cosinor method we were able to show the persistence of a circadian rhythm in the motor activity of diabetic rats, but with a mesor and amplitude lower than in controls (amplitude rested at 60+ of the mean level) and its acrophase advanced to 0148 hr.

The metabolic activity pattern of diabetic rats also changed: whereas controls showed a greater metabolic activity during the night (70+ food; 82+ water; 54+ urine; 67+ faeces), diabetics did not show differences between both phases of the LD cycle. Water ingested and urine excreted by the diabetic group were higher than normal during light and dark periods; food consumed and faeces excreted were higher than controls only in the light phase.

These data suggest that alterations in circadian rhythms of plasma corticosterone and motor activity are consecutive to the loss of the feeding circadian pattern, due to polyphagia and polydipsia showed by these animals, which need to extend intakes during the light and dark phases.     

The circadian clock as a molecular calendar

There are two dominant environmental oscillators shaping the living conditions of our world: the day-night cycle and the succession of the seasons. Organisms have adapted to these by evolving internal clocks to anticipate these variations. An orchestra of finely tuned peripheral clocks slaved to the master pacemaker of the suprachiasmatic nuclei (SCN) synchronizes the body to the daily 24h cycle. However, this circadian clockwork closely interacts with the seasonal time-teller.

Recent experiments indeed show that photoperiod—the dominant Zeitgeber of the circannual clock—might be deciphered by the organism using the tools of the circadian clock itself. From the SCN, the photoperiodic signal is transferred to the pineal where it is decoded as a varying secretion of melatonin.

Different models have been proposed to explain the mechanism by which the circadian clock measures day-length. Recent work using mutant mice suggests a set of two molecular oscillators tracking dusk and dawn, respectively, thereby translating day-length to the body. However, not every aspect of photoperiodism is covered by this theory and major adjustments will need to be made to establish a widely acceptable uniform model of circadian/circannual timekeeping.     

A Comparative Study of Several Samples of Neutral Rep and Their Effects on Paramoecium Caudatum

Several samples of neutral red have been studied; first, for the determination of their relative color intensities, and second, to determine their toxic effects on Paramoecium caudatum.

It was found that each of these dyes varied in physical characters both in dry form and in solution. The duration of life of groups of Paramoecia varied in each dye solution. These differences are probably due, at least in part, to differences in the chemical composition of each dye. This suggests that the varying results obtained by experimenters, in staining living material when using the same kind of dye under uniform conditions, may probably be attributed to these differences of chemical constitution of the dyes.

In the comparison of the toxic effects of the several samples most uniform results were obtained at concentrations of 1:5,000 and 1:10-000. At a concentration of 1:100,000 neutral red was found toxic but the results obtained were not in agreement with those obtained at lower concentrations.     

Evaluation of high sensitive C-reactive protein and 5-10 methylenetetrahydrofolate reductase genotype in Japanese young adults

Primary objective: To carry out a preliminary evaluation of subclinical inflammation and its genetic background in young adults.

Research design: Fifty-five healthy Japanese young adults aged 19-27 years (37 males and 18 females, mean age: 22.3 years), and 58 healthy Japanese adults aged 40 to 60 years (21 males and 37 females, mean age: 51.5 years) were included in this study.

Methods and procedures: We measured plasma high-sensitive C-reactive protein (hs-CRP) levels and screened for the C677T polymorphism of the 5-10 methylenetetrahydrofolate reductase gene (MTHFR), which is considered a genetic risk factor for atherosclerosis, by HinfI digestion.

Main outcomes and results: Hs-CRP levels of the young adult group were significantly lower than the levels of the middle aged group (0.014±0.030 mg/dl vs. 0.031±0.040 mg/dl, p=0.005). The levels were significantly higher in males than in females (0.028±0.019 mg/dl vs. 0.013±0.010 mg/dl, p=0.008) among young adults. Furthermore, we evaluated the relationship of the C677T genotype and hs-CRP values, but found no association between them.

Conclusions: Although the sample size is limited, our preliminary study demonstrated the profiles of hs-CRP in Japanese young adults. Further investigation will be needed to establish the guidelines for customized school health education using sensitive laboratory and genetic markers.     

A Discussion of Some of the Factors Causing Variable Results with Flagella Stains

Most flagella stains would probably give more consistent results if they were of stable composition and more was known of the factors influencing the mechanism of their action. Many of the mordants that have been recommended change rapidly, both chemically and physically, after preparation.

The reaction of some mordants, such as that of Loeffler, seems to be an important factor in their use. It is complicated, however, by temperature effects, oxidizing and reducing agents, the age of the bacterial cultures and variations in different species.

Users of flagella stains will probably always have to select or alter methods to suit the cultures to be stained. Any method which makes use of a minimum of complicated solutions and operations is most likely to give consistently good results.

No clearing or decolorizing agent tried changed poor preparations to good ones, but sometimes good ones can be improved by this treatment.     

Regression Models for the Estimation of Orcadian Rhythms in the Presence of Effects Due to Masking

The estimation of human circadian rhythms from experimental data is complicated by the presence of “masking” effects associated with the sleep-wake cycle. The observed rhythm may include a component due to masking, as well as the endogenous component linked to a circadian pacemaker. In situations where the relationship between the sleep-wake cycle and the circadian rhythm is not constant, it may be possible to obtain individual estimates of these two components, but methods commonly used for the estimation of circadian rhythms, such as the cosinor analysis, spectral analysis, average waveforms and complex demodulation, have not generally been adapted to identify the modulations that arise from masking. The estimates relate to the observed rhythms, and the amplitudes and acrophases do not necessarily refer to the endogenous rhythm.

In this paper methods are discussed for the separation of circadian and masking effects using regression models that incorporate a sinusoidal circadian variation together with functions of time since sleep and time during sleep. The basic model can be extended to include a time-varying circadian rhythm and estimates are available for the amplitude and phase at a given time, together with their joint confidence intervals and tests for changes in amplitude and acrophase between any two selected times. Modifications of these procedures are discussed to allow for non-sinusoidal circadian rhythms, non-additivity of the circadian and time-since-sleep effects and the breakdown of the usual assumptions concerning the residual errors.

This approach enables systematic masking effects associated with the sleep-wake cycle to be separated from the circadian rhythm, and it has applications to the analysis of data from experiments where the sleep-wake cycle is not synchronized with the circadian rhythm, for example after time-zone transitions or during irregular schedules of work and rest.     

Masking in Humans: The Problem and Some Attempts to Solve IT

Different types of masking are discussed together with an account of the masking effect that the sleep-wake cycle exerts upon the circadian rhythms of body temperature and urinary excretion. The relative importance to masking of the several components of differences between sleeping and wakefulness are then assessed.

Means to deal with the problem of masking fall into two major categories. These attempt to minimise masking effects by protocols such as constant routines or control days, and mathematical models which separate results obtained in the presence of masking influences into endogenous and exogenous components. (The problem of the extent to which masking influences can render the endogenous component of a rhythm an impure reflection of the internal oscillator is considered also.) These different techniques are compared with respect to their usefulness and assumptions.

Finally, a brief speculation is given of the usefulness of masking.     

The use of Constant Routines in Unmasking the Endogenous Component of Human Circadian Rhythms

A major problem in the study of the internal clock(s) that drives human circadian rhythms is that due to the effect produced by rhythmicity of habits and external influences ('masking'). A particularly potent factor in this respect is the sleep-wake cycle. It is anomalous that, even though this masking influence is widely accepted, most studies of circadian rhythmicity have been performed in the presence of such interferences.

A protocol is described, the constant routine, by which these exogenous influences can be minimized, thereby enabling a closer scrutiny of the internal clock(s) to be made. An account is given of the different circumstances in which the constant routines have been used together with the results derived from such studies. Briefly, they indicate that nychthemeral studies can give misleading information about the rate of adjustment of the internal clock to various manipulations, e.g. time-zone transition, shift work.

In addition, future studies making use of constant routines are described, in particular those which might enable the presence of more than one internal clock to be established.     

The internal synchronization of five circadian functions of the rabbit

Five different physiological functions of the rabbit (hard faeces and urine excretion, food and water intake and locomotor activity) were registered during LD 12:12 and during continuous light conditions (LL).

(1) In LD 12:12 a strong synchronization of the five parameters existed. The minima of all functions consistently occurred during the hours of light. The nocturnal percentage of overall 24-hr events was increased significantly in 'hard faeces excretion' (66±8 (S.D.) %), 'water intake' (64±15 (S.D.) %) and 'urine excretion' (58±10 (S.D.) %). The nocturnal percentage of locomotor activity was significantly increased during the dark-hours in 9 out of 14 animals. In the other five individuals prominent peaks were present even during the photoperiod. On the average of all 14 animals 5S±13 (S.D.) % of the 24 hr events of locomotor activity occurred during the night. Despite a trough during the cessation of hard faeces excretion the events of food intake were not elevated significantly during the dark hours.

(2) During LL the synchronization of the five functions within each animal persisted during the complete 90-day LL period. A free-running circadian rhythm with-: = 24.8±0.5 (S.D.) hr was present in the four rabbits kept in LL conditions within 5-16 days after the withdrawal of the zeitgeber.

(3) In addition to the circadian period the power spectrum analysis of data obtained during LD 12:12 revealed significant ultradian periods of an average period length of 11,6 hr (hard faeces and urine excretion), 8 hr (food and water intake, locomotor activity) and 4 hr (food intake, locomotor activity). During the free-run ultradian periods of 8 and 3.2-4.2 hr were significant in almost all parameters.

(4) During LL the level of locomotor activity was reduced for 13±16 (S.D.) %, the events of food intake were increased for 17±12 (S.D.) %.

(5) The reinserted LD 12:12 zeitgeber re-entrained the circadian rhythms within 25-45 days.

(6) These results provided evidence of a predominant nocturnality of the rabbits under investigation.     

Comparative Studies on the Orcadian Rhythm of Corticosterone Lipid and Cholesterol Levels in Adrenals and Blood of Rats

The time-dependent relationship of corticosterone, lipids and cholesterol over a 48-hr period was studied in the adrenals and blood of rats. In addition an attempt was made to determine whether there was a reciprocal dependence among these compounds and also a correlation between corticosterone and cholesterol in the adrenals and blood.

Corticosterone and cholesterol exhibit a circadian rhythm in the adrenals and blood. The same is true for lipids in the serum. A reciprocal dependence between the compounds in the adrenals and blood could not be demonstrated. Only the time-dependent processes of the corticosterone content in the adrenals and plasma are well correlated with each other. High levels of these steroids in the adrenals are associated with high levels of these steroids in the plasma and vice versa.

An inverse correlation between corticosterone and cholesterol exists in the adrenals and in blood. Maximal levels of corticosterone correspond to minimal levels of cholesterol and vice versa.     

Reduction of DNA fragmentation and hydroxyl radical production by hyaluronic acid and chondroitin-4-sulphate in iron plus ascorbate-induced oxidative stress in fibroblast cultures

Glycosaminoglycans (GAGs), components of extracellular matrix, are thought to play important roles in cell proliferation and differentiation in the repair process of injured tissue. Oxidative stress is one of the most frequent causes of tissue and cell injury and the consequent lipid peroxidation is the main manifestation of free radical damage. It has been found to play an important role in the evolution of cell death. Since several reports have shown that hyaluronic acid (HYA) and chondroitin-4-sulphate (C4S) are able to inhibit lipid peroxidation during oxidative stress, We investigated the antioxidant capacity of these GAGs in reducing oxidative damage in fibroblast cultures.

Free radicals production was induced by the oxidizing system employing iron (Fe₂₊) plus ascorbate. We evaluated cell death, membrane lipid peroxidation, DNA damage, protein oxidation, hydroxyl radical (OH_•) generation and endogenous antioxidant depletion in human skin fibroblast cultures.

The exposition of fibroblasts to FeSO₄ and ascorbate caused inhibition of cell growth and cell death, increased OH_• production determined by the aromatic trap method; furthermore it caused DNA strand breaks and protein oxidation as shown by the DNA fragments analysis and protein carbonyl content, respectively. Moreover, it enhanced lipid peroxidation evaluated by the analysis of conjugated dienes (CD) and decreased antioxidant defenses assayed by means of measurement of superoxide dismutase (SOD) and catalase (CAT) activities.

When fibroblasts were treated with two different doses of HYA or C4S a protective effect, following oxidative stress induction, was shown. In fact these GAGs were able to limit cell death, reduced DNA fragmentation and protein oxidation, decreased OH_• generation, inhibited lipid peroxidation and improved antioxidant defenses.

Our results confirm the antioxidant activity of HYA and C4S and this could represent a useful step in the understanding of the exact role played by GAGs in living organisms.     

Circadian rhythms and sleep in human aging

This issue of Chronobiology International is dedicated to the age-related changes in circadian rhythms as they occur in humans. It seems timely to give an overview of the knowledge and hypotheses on these changes now that we enter a century in which the number and percentage of elderly in the population will be unprecedented. Although we should take care not to follow the current tendency to think of old age as a disease—ignoring the fine aspects of being old—there is definitely an age-related increase in the risk of a number of conditions that are at least uncomfortable.

Circadian rhythms have been attributed adaptive values that usually go unnoticed, but can surface painfully clear when derangements occur. Alterations in the regulation of circadian rhythms are thought to contribute to the symptoms of a number of conditions for which the risk is increased in old age (e.g., sleep disturbances, dementia, and depression). A multidisciplinary approach to investigate the mechanisms of age-related changes in circadian regulation eventually may result in treatment strategies that will improve the quality of life of the growing number of elderly.

Although diverse topics are addressed in this issue, the possible mechanisms by which a deranged circadian timing system may be involved in sleep disturbances receives the most attention. This seems appropriate in view of the numerous studies that have addressed this relation in the last decade and also because of the high frequency and strong impact of sleep disturbances in the elderly. This introduction to the special issue first briefly addresses the impact of disturbed sleep in the elderly to show that the development of therapeutic methods other than the currently available pharmacological treatments should be given high priority. I believe that chronobiological insights may play an important role in the development of rational therapeutical methods.(Chronobiology International, 17(3), 233-243, 2000)     

On the Role of Energy Metabolism in Neurospora Circadian Clock Function

Neurospora crassa (bdA) mycelia were kept in liquid culture. Without rhythmic conidiation the levels of adenine nucleotides undergo circadian changes in constant darkness. Maxima occur 12-17 hr and 33-35 hr after initiation of the rhythm, i.e., at CT 0-6 hr. Pulses of metabolic inhibitors such as vanadate (Na₃Vo₄), molybdate (Na₂MoO₄: 2 H₂O), N-ethylmaleimide (NEM), azide (NaN₃), cyanide (NaCN) and oligomycin phase shift the circadian conidiation rhythm of Neurospora crassa. Maximal advance phase shifts are observed at about CT 6 with all inhibitors.

Pulses of N,N'dicyclohexylcarbodiimide (DCCD) and light phase shift the conidiation rhythm following a phase response curve different from those of the other agents (maximal advance at about CT 18-24). The phase shifts with DCCD and light are significantly larger in the wild type compared to the mitochrondrial mutant poky. Such differences are not found in PRCs of the protein synthesis inhibitor cycloheximide.

[³¹P] NMR spectra of wild type Neurospora crassa and the clock mutants frq 1 and frq 7 which differ in their circadian period lengths did not reveal differences in the concentrations of adenine nucleotides, pyridine nucleotides or sugar phosphates. Starvation causes drastic changes of the levels of adenine nucleotides, phosphate and mobile polyphosphate without effecting phase or period length of the circadian rhythm.     

Rapid reset of the corticosterone rhythm by food presentation in rats under acircadian restricted feeding schedule

Corticosterone levels were determined in the 7-week-old male rat maintained under different feeding and lighting schedules. At 4 weeks of age, the animals were kept either under a natural photoperiod (LD) or were subjected to continuous illumination (LL). Access to food was either ad libitum or restricted to an 8 hr span per 24 hr (circadian) or 32 hr (acircadian).

The food signal seemed able to synchronize the corticosterone rhythm to its own circadian periodicity, irrespective of the lighting regimen. No synchronization was observed in serially sampled LL or LD rats under an acircadian feeding schedule. Instead, the group acrophase appeared 24 hr subsequent to food presentation. Regarding individual patterns, many rats showed an acrophase or a peak also at that time. We speculate that an endogenous circadian mechanism was reset by the food signal, whenever it appeared.     

The Relative Zeitgeber Strength of Lights-On and Lights-Off Is Changed in Old Mice

The daily activity pattern of old mice is characterized by a decreased amplitude, a phase advance, and less stable relationship between lights-off and the onset of the main activity maximum. When analyzing the possible causes of these changes, it must be remembered that the activity rhythm of laboratory mice is bimodal, with a main peak in the first half of the dark time and a secondary one shortly after lights-on. Thus it seems to be controlled by at least two circadian oscillators—an “evening oscillator” coupled more strongly to lights-off and a “morning oscillator” coupled to lights-on—though both oscillators are also coupled to each other. The objective of the present paper was to investigate the putative changes in the strength of these couplings in HaZ:ICR mice of different ages (adult animals of 20 weeks, n=12; old mice of 72 and 91 weeks of age, n=6 each) and kept in a 24h LD-cycle with a gradually reduced light:dark ratio.

In adult mice, lengthening the dark time caused the onset of the main maximum of activity to be delayed in relation to the time of lights-off, while the morning maximum of activity was advanced in relation to lights-on. On average, the sizes of the advance and the delay were equal. As a consequence, the activity pattern did not shift in relation to the middle of the dark time. Lengthening the dark time resulted in a bigger (on average, 1.5h) difference between the evening and morning activity onsets. Under short photoperiods (≤2h of light) the activity rhythm started to free run, and the difference between evening and morning activity onsets decreased again. The changes obtained in senile mice were similar. However, the limits of entrainment were reached with longer photoperiods compared to adult animals. Also, the phase delay of the activity onset in the evening was much less, nearly zero. As a consequence, the activity pattern as a whole phase-advanced in relation to the middle of the dark time.

A model was proposed in which lights-off triggers advances of the “evening oscillator,” lights-on delays the “morning oscillator,” and the two oscillators are coupled with each other. Though it was probably the case, decreased coupling strengths could not be shown with the present experimental approach. However, it was clearly evident that, with increasing age, the advancing effect of lights-off exceeded the delaying effect of lights-on.     

Circadian Rhythms of Plasma Renin Activity and Aldosterone: Changes Related to Age, Sex, Recumbency and Sodium Restriction. Chronobiologic Specification for Reference Values

Plasma renin activity (PRA) and aldosterone (PA) levels are characterized by a circadian rhythmicity (CR). The present study revealed that this rhythmicity is influenced by several factors including posture, sodium intake and age. Time-qualified PRA and PA reference intervals can reduce the incidence of false positives and false negatives in a diagnostic work-up. The circadian rhythmicity of PRA and PA have been quantified in relation to posture, sodium intake and age. The cosinor procedure has been applied to quantify the properties of the circadian rhythmicity under these conditions.

Chronograms and circadian parameters can be used to optimize the use of PRA and PA measurements in clinical practice. The chronobiological specification of reference values for PRA and PA is of valuable importance since the assessment of PRA and PA circadian rhythmicity has a diagnostic interest for a certain type of clinical disorder. It should be noted that several studies have described circannual variations for renin and aldosterone. The next step in the optimation of laboratory time-qualified reference values is the assessment of changes induced by the deterministic factors on a circannual domain.     

Micro-Manipulation of Cells in Mammals

An arrangement of apparatus for applying micro-manipulative procedures to cells in living mammals is described. It was found satisfactory for manipulation of pericapillary cells and capillary endothelium in the greater omentum of the cat.

An animal board, a Bausch and Lomb triple purpose micro-projector and a double Fitz micro-manipulator were mounted on a spring platform. The micro-needles were drawn from Pyrex rods by hand. The stage of the microscope was modified to protect the condenser from fluids. Warm saline solution was carried to the exposed omentum thru flexible rubber tubing.

The use of a micro-dissection chamber which immobilized the part of the omentum under manipulation is explained. The construction of this chamber is shown by diagrams. A camera support was bolted to the base of the micro-manipulator.

The arrangement of apparatus is shown by a photograph.     

Enhanced Superoxide Production with on Change of the Antioxidant Activity in Gingival Fluid of Patients with Chronic Adult Periodontitis

In the gingival crevicular fluid (GCF) of control and chronic adult periodontitis (CAP) patients there is a spontaneous release of O₂^- radicals from polymorphonuclear leukocytes (PMN). The addition of the exogenous stimuli phorbol myrystate acetate (PMA) decreased the O₂^- formation in control GCF, while in CAP patients produced a marked enhancement of O₂^- generation.

The circulating PMN of control subjects did not show a spontaneous O₂^- formation, differently from CAP patients. On the contrary, a similar O₂^- production was measured when the circulating PMN were stimulated with PMA.

Moreover, the antioxidant activity measured in 10μl of cell free gingival supernatant (GS) of control and CAP patients had the same values by inhibiting 12.6% and 18.9% respectively of the O₂^- formation supported by a xanthine/xanthine oxidase system.

Probably, the protective or destructive effect of PMN in GCF of CAP patients depends on the variations of the rate of O₂^- formation in respect to the intrinsic antioxidant property of GS.