Comparison of immunological and endocrinological markers associated with major depression.

Natural-killer-(NK)-cell activity and blood levels of interleukin 2 (IL-2), dehydro-epiandrosterone (DHEA), DHEA sulphate (DHEA-S) and cortisol were measured in 17 patients with major depression and 10 control subjects. Depression severity was evaluated using the Zung Self-rating Depression Scale. NK-cell activity and IL-2 levels were measured using a chromium-51 release test and an enzyme-linked immunosorbent assay, respectively. Radio-immunoassays were used to measure serum cortisol, DHEA and DHEA-S. As would be expected, patients with major depression had a higher score on the Zung Self-rating Depression Scale than healthy controls. Compared with controls, NK-cell activity and levels of cortisol and DHEA were reduced in patients with major depression, whereas IL-2 levels were increased. No difference was observed in DHEA-S levels between patients and controls. A reduction in NK-cell activity and DHEA levels, and an increase in IL-2 levels appear to be associated with major depression. Whether these changes are the cause or the consequence of the depression remains to be determined.

[Coping with stress in the elderly].

In terms of adaptability, unlike inanimate objects, living organisms exist in a dynamic balance between "wear and tear" and "repair and recovery". We regarded 17-hydroxycorticosteroids (17-OHCS) as a compound related to tissue "wear and tear" (Hans Selye) and sought for a compound related to tissue "repair and recovery". This led us to the discovery of 17-ketosteroid sulfates (17-KS-S) in urine. Elderly persons, unlike young adults, show low levels in 17-KS-S and little diurnal changes. In an elderly person 17-KS-S can decrease on significant life events (e.g. a spouse's death) and remain at low levels for a long time. Elderly persons with frailty need to improve their lifestyle (meals, exercise, rest, sleep, etc.) qualitatively and quantitatively to adapt themselves to stress adequately. Increased 17-KS-S levels were to be related to improvement in lifestyle.

[17-KS sulfate as a biomarker in health and disease].

We attempted to determine compounds in human urine which, differing from 17-OHCS, show high values in healthy individuals, decrease with failing health, clearly decline with advancing disease and finally reach very low values in severe disease. We have initially established that 17-ketosteroid sulfates (17-KS-S) are the compound we were searching for. Hans Selye regarded stress as the rate of wear and tear and 17-OHCS as its indicator, but we considered that, differing from inanimate objects, living organisms consume energy to cope with stress and "repair" "wear and tear" of the tissue "and recover" its function. This concept led us to determine the organism's requirements for two mechanisms: "Wear and tear" which could be represented by the secretion of 17-OHCS (Selye), and "repair and recovery", which could be determined by the amounts of 17-KS-S (Nishikaze) derived from dehydroepiandrosterone sulfate (DHEA-S), a product of the adrenal cortex, which enhances neuronal survival, long-term memory, maintains the function of peripheral tissues (skin, bone-muscle, gastro-intestinal and vascular system etc.), increases lipid metabolism, protein anabolism (antiobesity, antiatherosclerosis etc.) and stimulates the immune system. We hold that the stress response is a series of biological processes, beginning with CRH.ACTH and Catecholamines, Cortisol (17-OHCS), followed by Insulin, acting as an anabolic agent and finally DHEA-S (17-KS-S) induces tissue repair utilizing the energy produced. Low levels of urinary 17-KS-S are observed not only in cases of low cortisol or catecholamines secretion, but also when these are elevated. Normalization of these levels is followed by an increase in 17-KS-S excretion. Balanced changes of hormones, such as 17-KS-S, 17-OHCS, Cortisol, Catecholamines and Insulin are seen in a normal organism under healthy lifestyles and disruption of the balance induces an apparent reduction in 17-KS-S. From this perspective, we hope to research the interrelations between biologically antagonistic 17-KS-S and 17-OHCS as well as other agents, focusing particularly on 17-KS-S, which represents a contact point for mind and body, which is kept in healthy balance by appropriate sleeping and exercise programs along with adequate food intake.

[17-KS sulfate as a biomarker in psychosocial stress].

We found an association between low levels of 17-Ketosteroid Sulfates (17-KS-S) in subjects under psychosocial stress. Stress associated with overwork and lack of sleep resulted in decreased levels of 17-KS-S and an increase in 17-Hydroxycorticosteroids (17-OHCS) levels. Alleviation of stress condition was associated with a restoration of the ratio of 17-KS-S/17-OHCS resulting from a slow increase in 17-KS-S and a decrease in 17-OHCS. In subjects with severe mental stress the ratio of 17-KS-S/17-OHCS showed markedly reduced values with a transient marked increase in 17-OHCS. There was a decrease in the levels of 17-KS-S in depressives, which was more pronounced during severe depression. There was no significant difference in 17-OHCS levels. In a cohort experiencing bereavement, there was a reduction in the levels of 17-KS-S, which remained low for about 50 days. With time, the level of 17-KS-S returned to the basal level established prior to spousal death. In this case, there was no significant change in the levels of 17-OHCS. Persistent severe stress over several months in a physician, led to a Herpes Zoster outbreak and Arrhythmia, resulting in hospital admission. The levels of 17-KS-S gradually decreased over this time reaching 1/3 of the normal baseline value, while 17-OHCS levels remained within normal ranges. These results suggest that measurement of 17-KS-S is indispensable for current research on psychosocial stress.

[17-ketosteroid sulfates and its application to kampo-medicine].

Traditional oriental medicine tells us that everything is interrelated and consists of combinations of opposing factors, "Yin-Yang". So we need to evaluate the normal state and pathologic condition from the perspective of dynamic hormone balance between cortisol and DHEA which are opposing and physiologically very important. Cortisol has a more functional side and is considered Yang, while DHEA has a more material side and is considered Yin. We tried to explain the concepts of KAMPO which include SHO (Yin-Yang. Excess-Deficiency), the 6 Stages of Disease and Deficiency of the Kidney, by measuring urinary 17-OHCS and 17-KS-S, metabolites of cortisol and DHEA. We think our effort may develop a good objective indicator of KAMPO's diagnosis and be useful for selecting of treatment.

[Stress and anticortisols--17-ketosteroid sulfate conjugate as a biomarker in tissue repair and recovery].

We attempted to determine compounds in human urine which, differing from cortisol (17-OHCS), show high values in healthy individuals, decrease with failing health, clearly decline with advancing disease and finally reach very low values in severe disease. We have eventually established that 17-ketosteroid sulfate conjugates (17-KS-S) are the compounds we were searching for. Hans Selye regarded stress as the rate of wear and tear and 17-OHCS as its indicator, but we considered that, differing from inanimate objects, living organisms consume energy to cope with stress and "repair" "wear and tear" of the tissue and "recover" its function. This concept led us to determine the organism's requirement for two mechanisms: "Wear and tear" which could be represented by the secretion of 17-OHCS (Selye), and "repair and recovery", which could be determined by the amounts of 17-KS-S derived from dehydroepiandrosterone (DHEA)-sulfate, a product of the adrenal cortex, which enhances neuronal survival, longterm memory, maintains the function of peripheral tissues, and stimulates immune system (17-KS mainly consists of 17-KS-S and 17-KS glucuronides 17-KS-G, the latter derived from cortisol, DHEA and testosterone). We hold that stress response is a series of biological process, beginning from CRH.ACTH and catecholamines, cortisol (17-OHCS), followed by insulin, acting as an anabolic agent and finally DHEA (17-KS-S) leads the tissues to repair by utilizing produced energy. Balanced changes of hormones, such as 17-KS-S, 17-OHCS, cortisol, catecholamines and insulin are seen in healthy individuals under healthy lifestyles and disruption of the balance brings apparent reduction in 17-KS-S. From this standpoint, we wish to develop research in interrelations between biologically antagonistic 17-KS-S and 17-OHCS, focusing particularly at 17-KS-S, which represents a contact point for mind and body, the healthy state of which is kept by appropriate sleeping and exercise programs on the basis of adequate food intake. The above three factors, 17-KS-S, 17-OHCS, and 17-KS-G, are expressed in creatinine ratio (mg/g creatinine).

[Stress and adaptation in humans--aging, illness, psychosocial stress].

We conducted research to determine compounds in human urine which show high values in healthy individuals, decrease with failing health (or bodily decay), clearly decline with advancing illness and finally reach very low values during terminal and severe illness. We have initially established that 17-ketosteroid sulfates (17-KS-S) are the substances we were searching for. Hans Selye regarded stress as the rate of wear and tear and 17-OHCS (cortisol) as its indicator. He further considered that stress is distinct from aging: There is no difference in the stress of ordinary life between young and old people, and there is no difference in 17-OHCS levels between the two. Aging is, however, accompanied by decreased adaptability. In terms of adaptability, we consider that, different from inanimate objects, energy transforming live organisms exist in a dynamic balance between "wear and tear" and "repair and recovery". This shows the need to determine the organism's adaptability to at least these two factors. We regarded 17-OHCS as a compound related to tissue "wear and tear" and searched for a compound related to tissue "repair and recovery", leading us to an identification of 17-KS-S. In other words, we have been considering the relation between "repair and recovery" and "wear and tear". The measurement of 17-KS-S which decreases with failing health, when combined with 17-OHCS, may make it possible to evaluate distortion of adaptation in a Yin-Yang like fashion, which can demonstrate the presence of illness, susceptibility to illness and progress of disease in individuals. It further adds a new dimension to clinical diagnostic acumen in an objective evaluation of psychosocial stress where results of routine clinical tests are often within normal limits, thus facilitating the monitoring of health and illness.

[Distortion of adaptation--wear & tear and repair & recovery--17-KS-sulfates and stress in humans].

We have conducted research to determine the compounds in human urine which have high values in healthy individuals, decrease with failing health (or bodily decay) and decline rapidly with advancing illness to reach very low values in severe illnesses. We initially established that 17-ketosteroid sulfates (17-KS-S) have the highest values in healthy individuals. Stress was regarded by Hans Selye as the rate of wear and tear and 17-OHCS as its indicator, but we considered that, different from inanimate things, energy transforming live organisms exist in a dynamic balance between "wear and tear" and "repair and recovery". Here it becomes important to determine the organism's adaptability at least to two factors. We regarded 17-OHCS as a compound related to tissue "wear and tear", and a search for a compound related to "repair and recovery", led us to the identification of 17-KS-S. In other words, we considered the relation between "repair and recovery" and "wear and tear". The measurement of 17-KS-S, which decreases with failing health, when combined with 17-OHCS, may make it possible to evaluate distortions in the organism (disturbance of adaptability), which bring the presence of illness or the susceptibility to illness in each person to our notice. Further adding a new dimension to clinical diagnostic acumen in an objective evaluation of psychosocial stress (emotional imbalance, dejection, depression, etc. due to the burden of a heavy debt, dismissal, urgent mental work against a deadline, transfer, inter-personal friction, parent-child relationship, etc.) where results of clinical routine tests are often within normal limits, and in the monitoring of health and illness.

[Considerations in clinical testing--implications of urinary 17-ketosteroid sulfate and 17-hydroxycorticosteroid measurements for stress research].

With the recent increase in number of patients with stress, the search is continuing for health-indicators that can be used to determine stress reliably and quickly. These indicators rely on subjective evaluations, and the establishment of an objective method of determining stress is necessary. We have developed a method involving the determination of the 17-Ketosteroid sulfates (17-KS-S) which has proved valuable for stress research. By simultaneous measurement of both 17-KS-S and 17-hydroxycorticosteroids (17-OHCS), which have different biological significance, and which originate from the adrenal gland, an organic Yin-Yang evaluation of the adrenocortical function can be made. The two values (mainly the 17-KS-S values) are closely correlated to the biological stress state as evaluated from psychosocial factors. This method should prove to be valuable in the clinic as an objective method of determining stress.

[Primary care considerations--17-ketosteroid sulfate-17-hydroxycorticosteroid method as a clinical stress indicator].

In primary care, the management of stress-related diseases occupies a wide spectrum of prevention and treatment, from holistic health care to final cure, centered on medical health and an organic integration of body, mind, and social parameters. Inquiries are being made at examinations and interviews to obtain information on the health condition of the patient, but, they are inadequate. There is a need for an objective indicator to help in the diagnosis. With the introduction of the Stress Barometer (measurements of 17-ketosteroid sulfates and 17-hydroxy-corticosteroids) it has become possible to obtain critical information on a patient in a very short time. This method allows early detection of stress, depression state and tiredness from work which is not presently possible from blood tests. It then becomes possible to call the patient's attention to these matters and to work at increasing awareness, energy levels, and responses to the conditions that are determined in this manner.

Direct determination of sulfate conjugates of 17-oxosteroids in urine by liquid chromatography.

We describe a new liquid-chromatographic method with fluorescence detection for direct simultaneous determination of four urinary sulfate conjugates of 17-oxosteroids (17OS), without use of solvolysis or enzymatic hydrolysis. The 17OS sulfates in urine samples are extracted with benzene as ion pairs in the presence of benzyltributylammonium chloride. These ion pairs are converted to hydrazones by use of dansyl hydrazine in trichloroacetic acid solution and then are separated by liquid chromatography. The proposed method, being simple and rapid, shows a good separation of 17OS sulfates and superior sensitivity, sufficient recovery, and good reproducibility.

Management of inflammation: the parametrical system, its practical and methodological position.

Since individual cases of inflammation differ one from another, and it is necessary to distinguish between adequate and inadequate inflammatory responses, the proper Management of Inflammation demands the simultaneous assessment of the various component processes. The parameters chosen for such diagnosis should be of broad specificity and show unequivocal and consistent changes wherever the inflammatory response is inadequate. Our long-term studies of carrageenin-induced inflammation in variously conditioned rats have led to a singling out of a set of parameters which satisfy the above three conditions, some fully and some partly: lipid peroxide, proamidase and lysosomal enzymes in exudate, sialoglycoproteins in serum and alkaline phosphatase, elastase in granuloma pouch tissue. Use of this system affords a broad reliable observation of inflammatory processes which an interpretation of isolated components alone could not provide, and determines the position and significance of other components in the inflammatory system. Moreover, it permits the discovery of correlations which would hardly be realized if single mechanisms were considered, and helps to form new hypotheses and to outline fields for further study.

Glandular kallikrein content in tissues of diabetic and hypertensive rats measured by enzyme immunoassay.

The contents of glandular kallikrein in the submaxillary gland and pancreas of normal, diabetic and hypertensive rats were compared using a specific enzyme immunoassay. The kallikrein levels in the submaxillary gland and pancreas of the diabetic rats were significantly lower than those of normal rats. On the other hand, the submaxillary and pancreatic levels in hypertensive rats tended to be higher than in normal rats.

Management of inflammation: lipid peroxide as a parameter for a better understanding of inflammatory processes.

The management of inflammation, based on an overall, coordinated observation of the inflammatory system, requires the assessment of the validity and biological significance of the various components in inflammation, a procedure that is more reliable than the interpretation of individual measurements in isolation. Such an overall approach, here developed from the model of carrageenin-induced inflammation, also predicates that lysosomal enzymes, lipid peroxide and proamidase (related, respectively, to the inflammatory response in a narrow sense, to tissue damage and to tissue repair) are three basic parameters required when studying inflammatory processes. This overall approach, moreover, shows that the inflammatory process must be regarded as being diachronic, and that in the light of the observation of exudate fluid it can be divided into three main stages, namely an early stage of exudation related to lipid peroxide, an intermediate stage of exudate dilution related to granuloma amidase, and a later stage of exudate concentration related to granuloma amidase and plasma protein.

Management of inflammation: proamidase as a parameter for a better understanding of inflammatory processes.

Coordinated observation of various factors in the inflammatory system and their relations shows that the levels of proamidase in granuloma pouch fluid in carrageenin-induced inflammation increase during the healing process whereas the proamidase shows (1) remarkably low values in rats injected with high doses of carrageenin, (2) low values in the earlier stages of inflammation with tissue damage (high values of fluid lipid peroxide and lactate dehydrogenase) and with phagocytosis (high values of fluid peroxidase and beta-glucuronidase), and (3) high values in the later stages where wound healing is progressing (high values of protein in plasma, fluid and granuloma pouch tissue and low values of fluid volume, fluid peroxidase, beta-glucuronidase and pouch tissue weight). In short, proamidase shows low values in the stage of tissue damage and constantly increasing values during the process of healing. Thus the measurement of proamidase, which reflects the degree of wound healing, provides an important parameter for a broad, coordinated observation of the inflammatory process, and may enable the management of inflammation.

Action of ionic iron in carrageenin-induced inflammation: aggravated inflammatory response with destruction of granuloma.

The experiments described lead to the conclusion that ionic iron enhances inflammatory responses with insufficient removal of carrageenin, and with insufficient granuloma formation probably due to a high degree of fibrinolysis. This non-adapted response may be due to the excessive production of lipid peroxide caused by ionic iron.

Streptozotocin diabetes: prolonged inflammatory response with delay in granuloma formation.

Inflammatory responses to carrageenin in streptozotocin-induced diabetic rats proved to be characterized by low values of granuloma tissue (tissue) weight and granuloma pouch fluid (fluid) volume, high values of fluid beta-glucuronidase (beta-Gl) and leucine aminopeptidase (LAP), and furthermore by high values of fluid lipid peroxide (LPO) and lactate dehydrogenase (LDH) and low values of fluid proamidase. These results in such diabetic rats were similar to those in rats injected with high doses of carrageenin except tissue weight and fluid volume. On the basis of measurements of three basic parameters required for coordinated observation of the inflammatory system, namely lysosomal enzymes, LPO, and proamidase (related respectively to the inflammatory response sensu stricto, to tissue damage, and to tissue repair), it can be concluded that the diabetic rats show an inadequately adapted inflammatory response, with decreased granuloma formation and reduced exudation, to noxious agents. This is probably due to a low sensitivity of the inflamed tissue in diabetic rats to noxious stimuli or chemical mediators, leading to a delay in the normal defence reaction of enhanced granuloma formation and exudation, thus resulting in a prolonged inflammatory response with a delay in wound healing.

Purification and characterization of rat low molecular weight kininogen.

Low molecular weight (LMW) kininogen was isolated from pooled rat plasma by chromatography on DEAE-Sephadex A-50, CM-Sephadex C-50, Blue-Sepharose CL-6B, and Sephadex G-100. It was shown to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoelectrophoresis. The molecular weight of rat LMW kininogen was determined to be 72,000 by SDS-PAGE. The LMW kininogen contained 83.5% protein, 4.0% hexose, 5.5% hexosamine, and 2.7% sialic acid. Kinin liberated from LMW kininogen by trypsin treatment was identified as an Ile-Ser-bradykinin(T-kinin) by analysis involving ion exchange column chromatography on CM-Sephadex C-25 and high performance liquid chromatography on a reverse-phase column (ODS-120T). LMW kininogen formed kinin with rat submaxillary gland kallikrein, but the kinin liberated was only 14% of the total kinin content, that is, that released by trypsin. In order to determine the immunochemical properties of LMW kininogen, specific antiserum was prepared in rabbits. The antiserum cross-reacted with high molecular weight (HMW) kininogen, but spur formation was observed between the LMW and HMW kininogens. The kininogen level in rat plasma was estimated to be 433 microgram/ml by a quantitative single radial immunodiffusion test.

Characterization of human salivary kallikrein: reactivities to human plasma kininogens and proteinase inhibitors.

In order to clarify the enzymatic properties of human salivary kallikrein, we investigated its actions on human plasma kininogens and response to proteinase inhibitors. Salivary kallikrein released kinin from both LMW and HMW kininogens, but acted preferentially on LMW kininogen. Salivary kallikrein did not completely release the kinin from HMW kininogen even after prolonged incubation (up to 60 min). The kinin releasing activity of salivary kallikrein was not inhibited by plasma proteinase inhibitors such as alpha 2M, alpha 2PI, AT III, and C1 INA after preincubation for 60 min at 37 degrees C, but was inhibited progressively by alpha 1AT as a function of preincubation time. SDS-PAGE revealed that concomitantly with the progressive inhibition a new band of 92,000 daltons, which seemed to be a complex of salivary kallikrein and alpha 1AT, was produced. The kininogen-depleted plasma inhibited the salivary kallikrein to the same degree as alpha 1AT. Therefore, it is suggested that alpha 1AT is the only inhibitor of salivary kallikrein in human plasma.