November 19661 NUTRITION REVIEWS 343

tion accounted for less than half of the reduction in ammonia excretion which fol- lowed injection of glutamate into the aci- dotic rats. The rnechanism(s) that brought about the remaining reduction in ammonia production is unknown. Although malate, alpha-ketoglutarate, and oxaloacetate in- hibit glutaminase activity, their concentra- tions in the kidneys were too small to play any role in the regulation of enzyme ac- tivity.

The glutamate levels in ra t kidneys de- crease within two hours after administra- tion of ammonium chloride (Goldstein and J. H. Copenhaver, Jr., Am. J. Physiol. 198, 227 (196'00)). Daily administration of am- monium chloride produces an increase in glutamate concentration in the kidney,

which by the third day approaches normal values (Goldstein, Zoc. cit.) , Daily adminis- tration of sodium bicarbonate produces a 25 per cent increase in kidney gIutamate concentration. This could account for a 10 to 15 per cent reduction in glutaminase ac- tivity. These observations suggest that the level of glutamate in the kidney probably plays a role in regulating glutaminase ac- tivity in acute acidotic states. The mecha- nisms operating in chronic acidosis and in alkalosis are unknown.

It should be emphasized that the reac- tions which lead to the changes in renal glutamate concentration are not known. This work does begin to emphasize the im- portance of glutamate and glutamine in maintaining a normal pH of body fluids.

STUDY OF ISOLATED SMALL INTESTINAL CELLS

Suspensions of intestinal epithelial cells prepared with buffered solutions o f hyaluronidase lake u p oxygen in linear fashion fo r about 40 minutes, and this uptake is increased when actively transported sugars are added to the media. These suspen- sions represent a significant advance in the study o f individual mucosal cells, the functional absorbing unit of the intestine.

Development of methods for the study of intestinal absorption and function has been of major importance to our knowledge of nutrition. Discoveries relating to these processes over the centuries reflect the his- tory of the mammalian sciences, and have been characterized by experimental tech- niques available during the corresponding period. I n the seventeent.h and eighteenth centuries, when investigations were largely limited to gross morphology, it was noted that the lymphatics were cloudy, and this cloudy appearance was attributed to fa t absorption.

Confirmation of this assumption came in the nineteenth century, when histological methods reached the necessary competence. Impetus for serious investigation of intesti- nal absorption came in 1864 with the work of Thiry in the laboratory of Karl Ludwig in Vienna. He surgically prepared blind

loops of intestine which opened onto the ab- dominal wall. By introducing test solutions into the loop and withdrawing samples, he was able to quantitate absorption as a func- tion of time in unanesthetized animals. Twenty-four years later, Vella in Bologna modified Thiry's method so that both ends of the isolated intestinal loop opened onto the abdominal wall, thereby permitting in- troduction of materials a t one end and their collection a t the other. H e also extended his techniques to the cecum and the colon.

Although these and similar techniques were significant contributions, they pro- vided Iittle insight into the mechanisms of absorption a t the cellular level. Background knowledge for the study of these phenom- ena began to accumulate during the last half of the nineteenth century as a consequence of advances in organic chemistry, physical chemistry, and enzymoIogy. Extensive con-

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troversies resulted. Some contended that in- testinal absorption could be explained by simple laws of diffusion while others main- tained that the primary phenomenon was osmosis. Then there were those who held firmly that active physiological forces had to be involved.

The protagonists of each point of view developed ingenious methods to seek evi- dence for their contentions. As a conse- quence, cannulation of intestinal lymphatics and the portal vein was developed to decide routes of absorption. Small laboratory spe- cies became important for quantitative measurements in isolated regions of the intestine or in the intact animal. These preparations in small animals had the ad- vantage of requiring less test material and permitting large numbers of analyses on whole intestinal tracts or entire carcasses. Much of our significant knowledge of the specificity of sugar absorption and the ab- sorption of fluids and electrolytes (T. H. Wilson, Intestinal Absorption. W . B. Saun- ders Co., Philadelphia, 1962) has been ac- quired in this way.

Advances in recent decades have again been the result of cross-fertilization be- tween different areas of science. A large measure of this has come from studies of cellular physiology by biochemically ori- ented investigators, who have introduced in vitro methods similar in type to those so valuable in many areas of biochemistry. Al- though isolated segments of raccoon in- testine were used by Jones as early as 1854, the first use of well-oxygenated prepara- tions of intestinal tissue was reported in 1949 by R. B. Fisher and D. S. Parsons (J. Physiol. 110, 36 (1949)), then working in a biochemistry laboratory. They applied their method to the study of amino acid and sugar absorption. Within the next few years, methods for studies of tissue accumulation of substances were developed as an exten- sion of this approach.

A method which has been responsible for

significant advances in the in vitro study of intestinal tissues has been the everted sac method of G. Wiseman and Wilson (Wilson and Wiseman,J. Physiol. 123,116 (1964)), which involves the use of small segments of intestine, turned inside out, filled with fluid and tied a t both ends. It provides the sig- nificant advantage of requiring the simplest of equipment. All that is needed is an in- cubation chamber such as an Erlenmeyer flask, a water bath, and a means of oxy- genation.

More recently, rings from intestine cut transversely in lengths of 1 to 5 mm. have been used in similar fashion to measure tissue accumulation of various substances. These techniques have been responsible for providing much of our recent quantitative knowledge on absorption and transport of amino acids and sugars. Even though the mucosa is responsible for a major part of the metabolic activity in the intestinal wall, i t is necessary to keep in mind that data obtained with the entire intestinal wall represent the composite activity of a vari- ety of cell types. Wilson has stated that the logical development of the above techniques should be use of the isolated intestinal cell, which is the functional absorbing unit of the intestinal epithelium.

Several attempts have been made to pre- pare suspensions of intestinal mucosal cells (D. S. Harrer, B. K. Stern, and R. W. Reilly, Nature 203, 319 (1964) ; K. C. Huang, Life Sci. 4,1201 (1965) ) . Recently i t has been shown that suspensions of cells can be obtained from the mucosa of the small intestine with buffered solutions of hyaluronidase (A. D. Perris, Canad. J . Bio- chem. 44, 687 (1966) ). First the small in- testine of the anesthetized rat is flushed in situ with 0.9 per cent NaCl and then re- moved. Thereupon it is everted, slightly distended with 0.9 per cent NaCI, and in- cubated for 12 minutes a t room temperature in 50 ml. of Krebs’ Ringer-phosphate saline containing 0.25 per cent fatty acid free bo-

November 1.9661 NUTRITION REVIEWS 345

vine serum albumin (BSA) and 0.15 per cent hyaluronidase (300 U.S.P. units per milligram),

The medium is pregassed with oxygen and the incubation is carried out in an atmos- phere of oxygen with gentle agitation. The cells are then washed twice at 4°C. with phosphate saline containing BSA. A typical preparation yields about 1 ml. of epithelial cells. The time required for preparation is about 35 minutes.

Studies in a Warburg apparatus have shown that such cells take up oxygen in linear fashion for up to 40 minutes with little if any additional uptake after 60 min- utes. The Qo, values (microlit.ers of oxygen consumed per milligram of cell protein per hour), calculated on the basis of the first 30 minutes, show that the presence of BSA is critical both in the preparative phase and during the time when oxygen consumption is measured. It is suggested that BSA binds free fatty acids which are toxic to metabo- lism and released by the cells during manip- ulation. No advantage appears to be gained by using concentrations of BSA greater than those stated above.

Histological studies of the tissues after treatment showed that all areas of the in- testine contribute cells, but that there is a wide variation in denudation between areas. Most of the tips of the villi and the epithe- lial cell layer above the necks of the villi were removed in the upper jejunum. In some cases the core of the villi was also re- moved. The mid-jejunum was less affected, and the ileum was little altered by the treat- ment, although strips of epithelium and whole villi were lost in occasional areas. Various methods of treating the suspensions and different histological stains gave con- vincing evidence that BSA protected against loss of nuclei and their characteristic stain- ing properties. I n agreement with other studies, the cell suspensions were found to contain about 83 per cent epithelial cells, the rest being predominately lymphocytes.

Although the author recognizes that other

media might prove superior to the phos- phate saline which was used, he did not find advantages in varying the media or using tissue culture media. The cell suspensions respired more actively in the presence of glucose than in its absence. Phbrhizin, known t o inhibit entry of glucose into the epithelial cell or to inhibit metabolism, re- duced oxygen uptakes to levels seen in the absence of glucose in the medium.

The intestinal cells provided by this method respire more actively than those ob- tained by other published methods. The author attributes this difference to the short time required for releasing the cells with hyaluronidase and their preparation for respiration studies. He suggests that cells released by trypsinixation (Harrer, Stern, and Reilly, Zoc. cit.) respire less actively in part, at least, because of the time required for preparation. The trypsinization method appears t o free a greater percentage of the mucosal cell population, including cells from the underlying tissue.

The hyaluronidase method appears to offer advantages for reliable measurement of concentration differences between cells and the incubation medium. All segments of the intestine contribute cells, but appar- ently not uniformly, as the histological studies reveal. It may therefore be neces- sary to treat different segments of the bowel accordingly. The cells of the epithe- lium migrate from crypts to villi during their life span and are extruded at the villus tips. Only those undergoing division take up thymidine avidly (G. G. Steel and L. F. Lamerton, Ezp. Cell Res. 37, 117 (1.96'6)).

It would be of interest to determine whether the mucosal cells represent a spec- trum of metabolic characteristics. This ap- pears to be the case with some of their other properties. If they are metabolically ho- mogenous regardless of their position along the villus or age since mitosis, future in- vestigations would be greatly simplified.

It is possible that techniques of this type

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may lead to selective isolation of certain outcome of these considerations, the science cell types. As an example, our knowledge of and practice of nutrition will benefit as the Paneth cells and argentaffine cells would techniques develop so that the properties of be significantly advanced if these types gastrointestinal tissues become more com- could be isolated for study. Whatever the pletely understood a t the cellular level.

CARBOHYDRATE METABOLISM OF RATS CONSUMING 450 P.B.M. FLUORIDE

Carbohydrate metaboliqm is altered in rats fed 460 p.p.m. fluoride. This may be an indirect e ject of these large amounts o f fluoride related to growth retardation and depressed food consumption.

The pros and cons of adding fluoride to water suppIies have been discussed rather vehemently in popular articles in recent years. Articles of more scientific intercst, dealing with the effect of fluorides on bio- chemical pathways, have recently appeared. Impairments in lipid metabolism, lowered fatty acid oxidase (Nutrition Beviews 18, 79 (1960) ) , have been discussed, as well as enhancement of fluoride toxicity by high levels of dietary fat.

E. J. Zebrowski and J. W. Suttie (J. Nu- trition 88, 367 (1966)) have recently in- vestigated the influence of large amounts of fluoride ingestion by rats on the metabolism of glucose Iabeled with GI4.

Holtzman (female) weanling rats were fed either a diet without fluoride or one con- taining 450 p.p.m. fluoride ad libitum for 30 to 35 days (preliminary) before adminis- tration of the labeled glucose. This large amount of fluoride caused a depression in growth during the preliminary period. The authors report 10,000 to 16,000 p.p.m. in the femur ash of animals fed fluoride as compared with 400 to 800 p.p.m. in the con- trol rats. Prior to administering the labeled glucose, the animals were fasted for 12 hours and then allowed food ad libitum for three hours.

Each rat was given 2.5 pc. of carrier-free g1~cose-UL-C~~ (12 pc. per pmole) intra- peritoneally. Carbon dioxide was collected at, 20 minute intervals for the first hour, 30

- minute intervals for the second hour, then a t 165 and 210 minutes.

For the determination of liver glycogen, the animals were stunned and exsangui- nated, and the livers immediately excised and frozen in dry ice until glycogen was iso- lated.

In the liver slice studies, livers were ex- cised, rinsed in Hastings medium I, and stored in the ice-cold medium until sliced and incubated. The authors state that slices from five a.nimals (control) were pooled and mixed, as were those from animals fed fluoride before slices were taken for incuba- tion. The reaction was stopped with NaOH.

In the glycogenolysis studies, liver slices were obtained from unfasted rats.

The results on expired carbon dioxide in- dicate that there was no difference between the control rats and those fed fluoride, al- though the specific activity of the CO:! of the rats fed fluoride was significantly higher (P < 0.01). Zebrowski and Suttie suggest ‘(. . . tha t the higher specific activity of the carbon dioxide expired by the fluoride-fed rats was the result of a lower expiration of carbon dioxide by the smaller fluoride-fed rats and not of an increased utilization of glucose for carbon dioxide production.”

In order to follow the rate of incorpora- tion of uniformly labeled g1uc0se-C~~ by ten fluoride and ten control rats, a time study was performed. Each rat was given 1.25 pc. of labeled glucose intraperitoneally ;