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This study was designed to determine whether or not a number of diverse rhythmic variables in the rat could be synchronized to meal timing. This was tested by restricting the availability of food; once during each 24-hour period an unrestricted quantity of food was made available for a 4-hour period to four different groups at different phases of the light-dark cycle, and the rhythms of the variables studied in the different groups were compared. Liver glycogen and serum glucose did synchronize to or were strongly influenced by feeding schedules; corticosterone and the several enzymes measured seemed to reflect an interaction of both the restricted feeding schedule and the light-dark cycle. The mitotic index in the corneal epithelium in all groups remained remarkably synchronized to the light-dark cycle and was altered only minimally by restricted meal timing. All groups on restricted feeding schedules gained less weight than the group fed ad libitum and maintained on a light-dark cycle. These studies caution against assuming that all body functions react in the same manner to different synchronizers; and they emphasize that one must not generalize about the synchronizing effect of meal-timing or even the light-dark cycle.

This study investigates, on a circadian basis, the variation in blood serotonin for a group of 64 healthy persons (volunteers of a Bundeswehr-Ausbildungskompanie); a pronounced circadian rhythm of 5-HT has been found. The variation in daily absolute levels is influenced by novelty stress; by this we refer to the stress resulting from initial contact between the person sampling blood and the patient. Obviously this diminishes as blood is repeatedly withdrawn from the same individual. Exposure to this situation results in an overall decrease in serotonin levels and a modification of the circadian pattern.

The alteration of extinctions of both FITC isomers by coupling to bovine serum albumin, human, and rabbit-gamma-globulin were studied. The extinction coefficients of these FTC conjugates were established. The emission characteristics of the FITC isomers and their conjugates were investigated under conditions normaly used in fluorescence microscopy. FITC I was found as the most suitable marker for human- and rabbit-gamma-globulin. In contrast the fluorescence of FTC II-BSA conjugates is stronger than that of the corresponding FTC I-BSA conjugate.

The efficiency of the membrane methods of Meijer (1972) and Lojda (1973) in histochemical lysosome tracing was studied and compared with the results of a preparation technique developed by us, employing fresh-frozen celloidin-coated sections; in addition, these methods were compared with results obtained with conventional formalin-sucrose-fixed livers. The lysosomes were traced by reactions for ACPase, beta-glucuronidase, arylesterase and acid-beta-galactosidase activity in rat livers systematically harvested at different time points of occurence of their maximal and minimal activities during the 24-h period, thus indicating a heterogeneity of lysosomes. 3. Optimal results with respect to the morphological appearance of lysosomes were obtained in fresh-frozen celloidin coated sections. This preparation method also caused no enzyme inhibition or loss, thus delivering comparatively the strongest reactions in livers and in enzyme models. 4. Day-time-dependent extralysosomal enzyme activities regularly occur in hepatocytes. Extralysosomal localizations however, are not a consequence of technically induced enzyme diffusion; they are best visualized in celloidin-coated sections; the membrane method produces less satisfactory results, and formalin-sucrose-fixed livers were least satisfactory.

To 4 male and 2 female subjects hexobarbital was applied orally at different times of the day: 2, 10, 18 h. Serum concentrations of hexobarbital were determined by HPLC procedure and the resulting pharmacokinetic parameters in a rotating iterative program. Application at 18 h resulted in a shorter invasion time, a higher maximum concentration and a delayed elimination; in addition, the AUC was elevated in relation to application at 2 or 10 h. In the 2 female subjects a different pattern was observed. The results lead to the assumption that application of hexobarbital is more effective in the evening than in the morning.

1. The circadian patterns of clinically relevant parameters were studied in 109 young, healthy soldiers. 2. Erythrocytes, hematocrit, creatinine and cortisol exhibited very stable rhythmic patterns which were found by continuous examinations of the same persons as well as in examinations of different persons at different times. 3. The rhythmic patterns of leukocytes, blood glucose and body temperature were strongly influenced by stress caused by the first confrontation with an unknown hospital-like situation or by pain caused by blood sampling.

For the determination of 5'-ribonucleotide phosphohydrolase (EC 3.1.3.5;5'-Nase) in rat liver, a radiochemical double-labelling assay was developed. [14C]-labelled AMP which is hydrolyzed to [14C]-adenosine by 5'-Nase activity is added to crude liver homogenates. After 30 min, the process is stopped and [2-3H]-adenosine added to estimate the loss of [14C]-adenosine during separation by ion exchange column chromatography. The enzymatic reaction was found to be linear in correlation with the enzyme content and the incubation time. The specificity of the reaction was evaluated by addition of beta-glycerophosphate which acts as a competitive inhibitor to eliminate the catalytic effect of non-specific phosphatases, and addition of alpha, beta-methylene adenosine 5'-diphosphate, a specific inhibitor of 5'-Nase; both cause an almost complete suppression of enzyme activity.

The influence of the mode of polymerization of GMA on the enzyme activities of differently fixed rat livers was studied. It was found that with increasing time of UV-polymerization several enzyme activities were inhibited whereas chemical polymerization was comparatively less harmful. The histochemical reactivity of substrates such as glycogen was not impaired by the mode of polymerization, which is in contrast to the increased basophilia of DNA and RNA after UV-polymerization.

Applying various preparations methods regarded as optimal for histochemical localization of enzymes in unfixed tissues, the following concordant results were obtained after demonstration of acid phosphatase, beta-glucuronidase and acid-beta-galactosidase activities in rat livers sampled at four typical time-points of a 24h-period (10:00, 16:00, 22:00, 04:00). 1. The lysosomes revealed with these marker enzymes differ with respect to their location within the liver lobules, their average number, size and reactivity during the 24h-period. 2. Extralysosomal activities also vary depending on the time of day. This is a normal and cytogenetically explicable event and not the consequence of an inadequate technique, and argues for a dual localization of lysosomal enzymes in the cell. 3. The chemical heterogeneity of the lysosomes and extralysosomal enzymes is obviously an expression of functionally different populations of lysosomes. In addition to the differences in location and average number, the variations in activity do not occur synchronously, but are phase-shifted.

The use of gaseous (DEPC, BOV) postfixation of frozen-dried tissues for the fixation of non-hormone proteins was systematically studied. The fixation ability was tested on the "protein droplet model" i.e. the protein droplets formed in mouse liver after intravenous injection of bovine serum albumin (BSA). 1. There is no difference in the fixation ability for BSA between DEPC and BQV. 2. Both fixatives yield a very poor fixation of BSA in frozen-dried livers. In consequence, this protein is already extracted during section mounting and incubation for immunofluorescent tracing causing a strong substantial loss. 3. Remaining protein traces, however, are still immunoreactive indicating that DEPC and BQV fixation does not affect the antigenicity in considerable extent.

The suitability of freeze-substitution in n-butanol and paraffin embedding of tissues for the histochemical demonstration of 5'-nucleotidase was investigated and compared with commonly used preparation techniques, such as fresh frozen sections and cryostate sections of cold formalin and glutaraldehyde-fixed rat liver. The influences of each step of the preparation techniques on the enzyme activity were controlled by a quantitative radiochemical assay. Freeze substitution was revealed to be superior to the commonly used preparation techniques with respect to: 1) high sensitivity and specificity of the histochemical 5'-nucleotidase reaction (this is based on the fact that incubation media with very low lead concentrations (0,6 mM/1) can be used); 2) excellent morphological appearance of the tissues showing cytological details of enzyme localization; 3) unlimited storage of the tissue materials and ease of sectioning.

1. The biochemically and histochemically determined glycogen rhythm was found to be shifted according to the shift of the 12: 12 h light-dark cycle. Deprivation of this synchronizer does not abolish the glycogen rhythm. 2. The circadian variations of the glycogen content are accompanied by a complete reorganization of the appearance of the hepatocyte inventory of cell organelles as seen in the analysis of the electron microscopical structure of liver cells. 3. The grade of the cytologically visible circadian changes is strongly correlated with the absolute glycogen content.

Circadian variations of hexobarbital oxidation rate in rat liver and the relationship to serum corticosterone levels were determined in 324 animals subjected to different lighting regimen and feeding schedules.--Rats maintained under standard light/dark conditions and with free access to food showed a significant circadian hexobarbital oxidase rhythmicity. Inversion of the light/dark cycle and time feeding was followed by significant changes of the 24-h means of hexobarbital oxidation rate as well as of the shape and phasing of the circadian pattern. The animals' response to the experimentally altered environmental conditions did not reveal a predictable relationship between the shape and phasing of the rhythmic patterns of liver hexobarbital oxidase activity and of corticosterone levels in blood serum; the rats subjected to timed feeding showed decreased levels of corticosterone whereas the hexobarbital oxidation rate of the liver was increased.

The influences of paraffin and GMA-embedding on acid phosphatase, esterase and beta-glucuronidase activity of differently fixed or freeze substituted rat livers were studied. 1. Embedding generally causes a reduction of the enzyme activities but improves considerably the quality of the microscopical pictures when compared with appropriate cryostat sections. Embedding therefore may serve as a very useful tool for detail studies on the cytological level. 2. The embedding media act differently on the reactive sites: a. Paraffin causes a heavy denaturation of the enzyme activity in lysosomes but preserves the activities of the ergastoplasmic (= "microsomal") enzymes. The degree of denaturation increases with increasing embedding temperature. b. GMA-embedding delivered opposite effects by preserving lysosomal activities and quenching endoplasmic enzymes. UV-polymerization of GMA causes a general inactivation of enzymes. 3. The histochemical reactivity of substrates such as glycogen was not influenced by the embedding. However, its most natural localization is achieved by freeze drying or isopropanol freeze substitution followed by GMA-embedding.