Robert E. Kuhlman

PHOSPHATASES IN EPIPHYSEAL CARTILAGE; THEIR POSSIBLE ROLE IN TISSUE SYNTHESIS.

An attempt is made to delineate three phosphatase systems in the epiphyseal mechanism of the rabbit, dog, and human being. Of particular interest is inorganic pyrophosphatase since it is present in substantial amount only in the proliferating cell zone. Some of its properties are described. An explanation of its functional significance to the growth mechanism is suggested.

The Biochemical Importance of the Hypertrophic Cartilage Cell Area to Enchondral Bone Formation

Quantitative microchemical estimations of multiple additional enzyme systems (hexokinase, inorganic pyrophosphatase, isocitric dehydrogenase, aldolase, phosphoglucoisomerase, and acid phosphatase) as well as repeat evaluation of alkaline phosphatase, glucose-6-phosphate, and lactic and malic dehydrogenase have been made in the individual structural areas of the epiphyseal plate of the developing rabbit epiphysis. These observations are related to quantitative estimation of glucose, lactate, adenosine triphosphate, phosphate, and hydroxyproline content in these areas. Large amounts of carbohydrate oxidative enzymes as well as substantial substrate and phosphatase content in the hypertrophic area of the epiphyseal plate indicate that this area of the epiphysis is not constituted of dead and dying cellular elements. These data support other tissue-culture and electron microscopic studies, which indicate this area to make a viable important cellular contribution to the process of enchondral bone formation.

A microchemical study of the developing epiphyseal plate.

Preliminary information of the general enzymatic characteristics of the normal stages of the development of endochondral bone in the dog and the effect of age are presented. The enzymes glucose-6-phosphate, lactic and malic dehydrogenase, and phosphoglucoisomerase are found to be active through the maturing stages of epiphyseal development. Alkaline-phosphatase activity, on the basis of acid-insoluble material comprising the area, is greater in the primary spongiosa than in other locations, as reported previously. The results are discussed in relation both to the total and the acid-soluble solid and phosphorus content in the areas. An over-all increase in the level of enzyme activity is noted in the epiphyseal plate as the newborn dog matures. The enzyme content in the epiphyseal plate is such as to indicate that the process of epiphyseal growth and bone formation is an enzymatically regulated sequence deriving energy and intermediates through the Embden-Meyerhoff pathway, the hexosemonophosphate shunt, and the tricarboxylic-acid cycle. Adaptations of microchemical techniques suitable for chemical investigation of biopsy material from diseased epiphyseal plates are presented.

Biochemical Investigations of Deer Antler Growth: Part I. Alterations of Deer Blood Chemistry Resulting from Antlerogenesis

A colony of thirteen male and female deer have been studied for seasonal fluctuation of blood calcium phosphorus, albumin, globulin, and alkaline phosphatase. The female animals had stable levels of these parameters of body chemistry throughout all periods of the years. The male animals, beginning i

The Biochemical Activity of Fracture Callus in Relation to Bone Production

Quantitative microchemical study of the tissues comprising fracture callus has been undertaken to correlate the biochemical activity of the bone repair process with its previously established morphological features. Areas of proliferating fibrous tissue, hypertrophic cartilage, new bone and undifferentiated granulation tissue were analyzed for their content of carbohydrate metabolizing and phosphatase enzymes. Fracture callus cartilage is biochemically similar to epiphyseal cartilage. Carbohydrate metabolism provides structural intermediates and energy for bone repair. Inorganic pyrophosphatase removes the inorganic pyrophosphate which accumulates from structural synthesis and prevents its inhibition of new bone calcification. The individual parts of the callus have identical biochemical function regardless of the age or healing time of the fracture callus.

Biochemical Investigations of Deer Antler Growth: Part Ii. Quantitative Microchemical Changes Associated With Antler Bone Formation

The localized content of lactic, malic, and glucose-6-phosphate dehydrogenases, phosphoglucoisomerase, alkaline phosphatase, and total phosphate at five selected stages of deer antler formation has been investigated by means of quantitative microchemical methods. Generally, the antler tine has a higher over-all level of enzyme activity than do the epiphyseal plates previously studied. Alkaline phosphatase in the preosseous zones of the tine rises to extremely high levels, much above anything reported in the epiphyseal plate. However, no increase in phosphate content was observed to be associated with the increase in alkaline phosphatase. The source of the seasonal elevation of circulating alkaline phosphatase occurring in male deer is shown in this paper, as well as in the previous paper of the series, to be the preosseous areas of the proliferating antler tine.

Enzyme alterations in the epiphyseal plate of the rabbit resulting from aminoacetonitrile administration.

Lathyritic lesions were induced in young growing rabbits by the administration of aminoacetonitrile. The animals with lathyritic lesions and those in a control group were sacrificed and frozen-dried sections were prepared from the epiphyseal plate of the femur after the method of Lowry. By use of specialized methods, the proliferating and hypertrophic cartilage-cell and primary-spongiosa areas of these animals were studied for their content of lactic, malic, and glucose-6-phosphate dehydrogenase, phosphoglucoisomerase, alkaline phosphate, and total phosphorus. Striking depression of lactic and glucose-6-phosphate dehydrogenase activity was observed in the areas of the hypertrophic cartilage cells and primary spongiosa. Phosphoglucoisomerase was reduced throughout all three areas. On the other hand, the content of total phosphorus, malic dehydrogenase, and alkaline phosphate in these areas was not disturbed. The most severe enzyme alterations occurred in those areas most disrupted by aminoacetonitrile. From these and previous observations, it is felt that the morphological disruption of the epiphyseal plate associated with aminoacetonitrile treatment results from the reduction in activity of several enzyme systems normally providing metabolic intermediates and high-energy phosphate necessary for tissue synthesis. These observations are discussed in relation to the nutritional status of the animals, previous studies of the developing epiphyseal plate of the dog, and the protective effects of thyroxine and triiodothyronine against aminoacetonitrile lesions.

The Biochemical Changes Preceding Tissue Death in Rats

An attempt has been made to delineate the immediate biochemical modifications which precede extremity death by analysis of glucose, adenosine triphosphate, phosphocreatine, lactate, and glycogen in certain of its tissues. Epiphyseal cartilage demonstrates considerable resistance to devitalization preserving apparent biochemical integrity beyond two hours. Skeletal muscle responds in a somewhat more active fashion. In general, epiphyseal cartilage and muscle demonstrate considerable tolerance to nutritional deprivation. Differences between epiphyseal cartilage and skeletal muscle in their biochemical response to devitalization are described.

Hexose-6-Phosphatase Activity in the Seminal Vesicles and Prostate Gland of the Rat.∗

Summary The seminal vesicles and prostate gland of the rat are capable of hydrolyzing both fructose-6-phosphate and glucose-6-phosphate. This hydrolytic activity is located in different regions from those containing acid and alkaline phosphatase. The activity toward the hexose phosphates is easily destroyed by fixing agents. The significance of the hexose phosphatase activity is briefly discussed in relation to the fructose metabolism of the male reproductive system.

Biochemical Biopsy of Skeletal System Lesions

Utilizing quantitative microchemical techniques developed by Lowry, an attempt was made to obtain biochemical biopsy specimens and to determine the enzyme content of morphologically selected areas from malignant and benign lesions of the skeletal system. Osteosarcoma, chondrosarcoma, proliferating bone from Pagets disease, and tissue from polyostotic fibrous dysplasia were analyzed for enzymes of carbohydrate metabolism, hexosamine, and total phosphate content. Generally the areas and lesions characterized by rapid proliferation were the most active enzymatically and in the selections from malignant neoplasms lactic dehydrogenase was predominant. Benign proliferating bone in Pagets disease was featured by more active aerobic pathways with more malic dehydrogenase. Comparison of neoplastic cartilage from the osteosarcoma with the morphologically similar cartilage from chondrosarcoma demonstrated considerable chemical differences between the two cartilage types. In general, the clinical course and manifestations of the lesions studied could be correlated with the metabolic activity as determined by the alkaline phosphatase and carbohydrate intermediate enzyme content.