Atomic absorption spectrophotometry applied to photographic densitometry.

For this study of photographic densitometry, sections of cartilage stained with Alcian Blue, safranin O and high iron diamine were photographed at x40 with Nikon photomicrography equipment on Kodak Panatomic X film with appropriate filters to enhance contrast. Portions of the developed negative films were selected from intercellular matrix regions, and circles of film equivalent in diameter to a 30-mu circle of tissue were obtained with a hand-held paper punch. Silver was eluted from the circles of film with 35% nitric acid, and the quantity of silver deposited on the film was determined by atomic absorption spectrophotometry as a measure of stain intensity. The intensity determined by this analytic procedure compared favorably with results obtained previously from the same tissue with microspectrophotometry. This method of silver analysis has advantages over earlier studies which used silver elution to determine photographic densitometry in its technical ease, accuracy and sensitivity. Furthermore, this method compares well with microspectrophotometry in its results and has the advantages of relative inexpensiveness and availability of equipment.

Hypercalcemia in association with a Leydig cell tumor in the rat: a model for tumor-induced hypercalcemia in man.

The etiology of tumor-induced hypercalcemia was investigated in a transplantable Leydig cell tumor of the Fischer rat. In this model, serum calcium rose from a baseline of 10.4 +/0 0.3 mg/dl to 12.5 + 0.4 mg/dl at day 10 and 16.4 +/- 1.3 mg/dl (p less than 0.001) at day 13 post transplant. Urinary calcium also increased from 1.52 +/- 0.17 mg/d to 3.52 + 0.72 mg/d (Day 12, p less than 0.01). Serum phosphate decreased from a baseline of 7.5 +/- 0.3 mg/dl to 5.5 +/- 0.6 mg/dl at day 13 (p less than 0.05). At day 13 serum immunoreactive parathyroid hormone levels fell 76% from baseline (p less than 0.01). Calcitonin increased from 59 +/- 2 pg/ml to 88 +/- 9 pg/ml (p less than 0.02). The plasma prostaglandin E metabolite, 13,14-dihydro-15-keto-PGE2 increased from 407 +/- 103 pg/ml to 647 +/-62 pg/ml (p less than 0.05) and the active Vit D compound 1,25(OH)2D increased from 94.8 +/- 5.2 pg/ml to 162.3 +/- 11.8 pg/ml (p less than 0.01). Urinary cyclic AMP did not decrease in parallel with the parathyroid hormone level and, in fact, increased from 146 +/- 3 nmol/d to 172 +/- 27 nmol/d (NS). Administration of the cyclooxygenase inhibitor indomethacin (20 mg/Kg/d) or hydrocortisone (50 mg/Kg/d) did not prevent the development of hypercalcemia. This model is similar to many patients with humoral hypercalcemia of malignancy who demonstrate suppression of parathyroid hormone with elevated urinary cyclic AMP excretion and may prove useful in the understanding of the responsible mechanisms.

Review of cancer among 4 religious sects: evidence that life-styles are distinctive sets of risk factors.

The occurrence of various types of cancer have been reviewed and evaluated in 4 religious groups. These patterns have been critically assessed in light of the distinctive life-style features of these groups. All 4 religious groups considered in this paper have reduced overall rates of cancer, suggesting that the life-styles of all 4 groups have merit in terms of reducing the overall risk of cancer. The rate of smoking among these groups is nearly nil, and the lung cancer rate in all 4 of these religious groups is strikingly low. Cancer of the oral structures, pharynx, larynx, and esophagus is also generally quite low. Amish and Hutterites have unusually high rates of breast cancer and juvenile leukemia. Reproductive factors frequently mentioned as risk factors for breast cancer cannot explain the excess breast cancer in the Amish and Hutterite women because they should have had the effect of reducing the rate. None of the numerous risk factors, normally suggested for leukemia, are consistent with this observation. The observations on ovarian cancer tend to confirm low parity and late age at first birth as risk factors, although the evidence is not entirely consistent. Also, contrary to common observations, the pattern of ovarian cancer contrasts greatly with the breast cancer pattern, suggesting dissimilar risk factors. Their low rate of cervical cancer is consistent with promiscuity being a strong risk factor, but other frequently suggested risk factors were generally inconsistent with the observations. Cancers of the stomach, colon, rectum, urinary bladder and prostate, in these 4 religious groups, are not readily explained by the risk factors commonly implicated in cancer of these sites. The patterns of a few types of cancers were consistent with the prevailing opinions of risk factors, but some cancers were poorly explained and, in some cases, the cancer patterns contradicted commonly held opinions concerning risk factors. Religions that provide strong directives for the personal lives of adherents result in distinctive life-style, reflecting multiple disease related factors (risk factors and protective factors). Disease related factors are related to each other in simple or more complex ways (e.g. additive, multiplicative or even more complex). Therefore, when dealing with distinctive life-styles, it may be unwarranted to attempt to isolate individual risk factors.

Spectral characteristics of metachromatically stained cartilage: effects of enzymatic degradation and dehydration.

When a microspectrophotometer was used to study cartilage, stained metachromatically with Azure A and Safranin O, the following 3 points were made: 1. the absorption maximum of stained cartilage matrix is identical to that reported for solutions of these dyes containing chondroitin sulfate; 2. partial enzymatic degradation of the cartilage matrix has no effect on the position of the absorption peak (although the intensity is greatly diminished); 3. ethanolic dehydration shifts the absorption peak somewhat toward the orthochromatic position, and decreases the intensity of the stain.

Enzyme histochemistry of undecalcified bone and cartilage embedded in glycol methacrylate.

Undecalcified bone and cartilage tissue blocks were fixed for 3 h in cold formol-calcium, rapidly dehydrated with a graded series of cold ethanol, and embedded in glycol methacrylate. 2 mum sections were produced with a Sorvall JB-4 microtome using glass knives. The quality of the sections were usually excellent except for hard bone from old subjects where the bone sometimes shattered while sectioning. This method is short, relatively uninvolved and eliminates en bloc decalcification. Moreover, the method is gentle enough to allow the histochemical demonstration of alkaline and acid phosphatase by the azo dye methods, and acid phosphatase, 5'-nucleotidase and ATPase by the lead precipitation methods.

The effect of short-term immobilization on the rabbit knee joint cartilage. A histochemical study.

Rabbit knee joints were immobilized in the extended position for 1 to 6 days, and examined by means of histochemical techniques. In the articular cartilage matrix, the earliest change was the loss of affinity for metachromatic stains, confirming earlier observations. The affinity of cartilage for high iron diamine and alcian blue stains was also diminished, although less dramatically; these stains demonstrated the concentration of glycosaminoglycans more accurately than metachromatic stains. With the colloidal iron method, changes were seen in the chondrocytes before there was any loss of affinity for metachromatic stains. Following only 1 or 2 days of immobilization, the chondrocytes in the gliding surface of the joint stained intensely with colloidal iron whereas those in the controls stained very minimally.

A hematoxylin and eosin-like stain for glycol methacrylate embedded tissue sections.

A staining procedure is described for use with glycol methacrylate embedded tissue sections which does not stain the plastic embedment or remove the sections from the glass slides. The basic dye is celestine blue B. It is prepared by treating 1 g of the dye with 0.5 ml concentrated sulfuric acid. It is then dissolved with the following solution. Add 14 ml glycerine to 100 ml 2.5% ferric ammonium sulfate and warm the solution to 50 C. Finally adjust the pH to 0.8 to 0.9 The acid staining solution consists of 0.075% ponceau de xylidine and 0.025% acid fuchsin in 10% acetic acid. Slides containing the dried plastic sections are immersed in the celestine blue solution for five minutes and in the ponceau-fuchsin solution for ten minutes with an intervening water rinse. After a final wash, the sections are air dried and coverslipped. This staining procedure colors the tissues nearly the same as hematoxylin and eosin procedures.

Histochemical investigation of adjuvant-induced arthritis.

Rats with adjuvant-induced arthritis were observed to have increased alkaline phosphatase, acid phosphatase (two isozymes), and ATPase activity in the radial zone of articular cartilage, at the osteochondral junction, and in the bone marrow elements. A qualitative and quantitative reduction azure A, PAS colloidal iron, alcian blud critical electrolyte concentration staining (0.4 and 0.9 M (mg Cl2) was also observed in corresponding areas. These findings suggest the degradation of the articular cartilage matrix with possible simultaneous or resultant calcification.

Leydig cell tumor induced hypercalcemia in the Fischer rat: morphometric and histochemical evidence for a humoral factor that activates osteoclasts.

A nonmetastatic, transplantable Leydig cell tumor of Fischer rats causes hypercalcemia in the host animals. The tumor's effects on long bones were studied with histochemical and morphometric methods. Small viable fragments of the tumor (about 1 cu mm) were transplanted into 59 Fischer rats. After 2 weeks, when the animals were sacrificed, the tumors weighed 13.9 +/- 4.8 gr (SD), and the animals had lost a mean of 10.1 g of weight. Compared with 16 normal control rats (mean weight gain of 9.5 g), the tumor-bearing rats showed: 1) a reduction in the volume of trabecular bone, which correlated well with the weight of the tumor; 2) a significant narrowing of the epiphyseal plate cartilage; 3) a diminution in the number of marrow fat cells, which in most tumor-bearing rats were totally absent; 4) an increase in the number of osteoclasts many of which were mononuclear and had to be identified with the aid of a histochemical reaction for acid phosphatase; 5) a great expansion of the marrow vascular channels that maintained an endothelial lining. Since there is no metastasis of the tumor cells, the effects of the tumor are presumably mediated by a humoral factor that activates existing osteoclasts and induces monocytes to differentiate into osteoclasts.

Degenerative changes in the digestive glands of rats bearing Leydig cell tumors.

We undertook this study to determine the effects of a transplantable Leydig cell tumor on the major digestive glands of the host rats. It has been known that after bearing this tumor for only two weeks, the rats become anorexic and cathectic, develop hypercalcemia and osteolysis, and their peripheral bone marrow becomes hyperplastic. We now demonstrate that the parotid glands undergo marked degeneration including depletion of secretory product, apparent loss of acinar organization and the appearance of conjoined nuclei. The submandibular and sublingual glands and the pancreas are virtually unaffected. The liver undergoes fatty degeneration, the Kupffer cells become more prominent and more numerous, and the sinusoids sometimes contain small islets of hemopoietic tissue.

Bone alkaline phosphatase kinetics studied by a new method.

Studies of enzyme kinetics by histochemical methods has been seriously impaired by available methods. This study combines two newer methods, i.e., sectioning undecalcified bone from glycol methacrylate and the use of atomic absorpton spectrophotometry for photographic densitometry. The results indicate that the substrate concentration for zero order kinetics of the alkaline phosphatase reaction in bone is 0.5 mg/ml of substrate; and the Km of the same reaction is estimated to be 0.25 mg/ml or 0.56 mM of substrate.