Stanley N. Gershoff

Vitamin B6 deficiency and oxalate nephrocalcinosis in the cat

Abstract Vitamin B 6 deficiency, characterized by failure of growth, emaciation, convulsions, anemia and oxalate nephrocalcinosis, has been produced in cats. The presence of large quantities of oxalate in the kidneys and urine of cats deficient in vitamin B 6 , in amounts sufficient to cause marked renal damage, is attributable to excessive endogenous formation of oxalate. The implications of these findings in relation to human disease states associated with oxalate are discussed.

Vitamin B6and Oxalate Metabolism

Publisher Summary There was considerable interest in the metabolism of oxalic acid by animals during the last half of the 19th century. Several years ago, it was observed that cats fed a commercial cat food developed oxalate nephrocalcinosis although there was no oxalate in their diet. Subsequent studies showed that because of the heat processing used in the preparation of the food, much of its vitamin B 6 content had become biologically unavailable. This chapter reviews some of the work that ensued because of these two observations. Deposition of oxalate in the urinary tract is a serious concomitant of several pathologic conditions of man. A relationship between vitamin B 6 deficiency and the endogenous production of oxalic acid has been observed in several animal species. Studies of the metabolism of oxalate precursors have raised new questions concerning the metabolism of a number of 2- and 3-carbon metabolites. It is now possible to produce in rats urinary calcium oxalate calculi that are similar to those seen in man. Thus, a new approach has been provided for studying the effect of diet on the etiology of urinary stone formation.

Effect of Vitamin B6 And Magnesium on Renal Deposition of Calcium Oxalate Induced by Ethylene Glycol Administration.

Summary Oral administration of ethylene glycol to rats and the resultant renal de-position of calcium oxalate were studied in terms of dietary levels of Vit. B6 and magnesium. Under the conditions of these experiments partial protection against oxalate deposition was achieved with excessive dietary Vit. B6 while complete protection resulted when large amounts of MgO were fed. The in vivo oxidation of ethylene glycol to CO2 was found to be related to amount of Vit. B6 administered. Dietary magnesium levels, however, did not affect oxidation of ethylene glycol and it is suggested that dietary magnesium protects against renal deposition of calcium oxalate by altering the solvent characteristics of urine.

Inhibitory Effects of “Bound” Insulin on Insulin Uptake by Isolated Tissues

“Bound” insulin preparations obtained from pooled human sera inhibited the uptake of crystalline insulin by isolated rat muscle (hemidiaphragm) and epididymal adipose tissue. When crystalline insulin alone was incubated with isolated tissues, its concentration in the incubating medium declined gradually, as shown by immunoassay of samples obtained at various intervals during the incubation. Addition of “bound” insulin preparations into the incubation medium greatly reduced the rate of disappearance of the crystalline insulin, “bound” insulin inhibiting the uptake of insulin by the tissues. The inhibition of insulin uptake by muscle, caused by “bound” insulin, was accompanied by inhibition of the biologic activity of insulin on this tissue. Preincubation of the isolated tissues with “bound” insulin, with whole fasting human sera from maturity-onset diabetics, or with crystalline insulin, prior to the addition of crystalline insulin, also resulted in significant inhibition of insulin uptake by these tissues. Synalbumin preparations obtained from fasting human sera by acid-ethanol extraction, as described by Vallance-Owen and collaborators, also inhibited the uptake of crystalline insulin by isolated muscle and adipose tissue and the biologic activity of insulin on muscle. Like “bound” insulin, the synalbumin extracts were inactive on isolated muscle and unreactive with anti-insulin antisera but exerted insulin-like activity on isolated adipose tissue. Their action when injected intraperitoneally into intact rats was similar to that of “bound” and crystalline insulin on the muscle and the adipose tissue. It is suggested that the synalbumin preparations obtained from sera by acid-ethanol extraction may contain the bulk of the serum “bound” insulin which contributes, at least in part, to the insulin inhibitory properties of these preparations.

Insulin Sensitivity in Vitamin B6-Deficient Rats

Increased insulin sensitivity of vitamin B6-deficient rat adipose tissue may be related in part to altered GH metabolism. The metabolism of glucose-U-14C in muscle and adipose tissue was determined in vivo as a function of insulin, GH and combined insulin-GH treatments. Vitamin B6-deficient rats were more sensitive to insulin and acute GH treatments, which exerted an insulin-like effect, than were ad libitum or pair-fed controls. Chronic GH treatment had an insulin-like effect on muscle and potentiated the action of insulin, while it inhibited the action of insulin in adipose tissue. The degree of response to chronic GH treatment was greatest in the vitamin B6-deficient rats. The data provide evidence that chronic GH treatment modified insulin sensitivity in vitamin B6-deficient rats.

The effect of fasting and refeeding on isolated rat adipose tissue activity in the presence of crystalline and “bound” insulin

Effects of crystalline and “bound” insulinobtained from human serum on net gas exchange and fat synthesis from glucose by isolated epididymal adipose tissue from fed, fasted and fasted, refed rats have been studied. Fasting resulted in a marked decrease in activity of epididymal adipose tissue in the presence of either crystalline or “bound” insulin. Refeeding rats after a 4 day fast resulted in hyperactivity of their adipose tissue to both forms of insulin which remained maximal after 7 days of repletion. Positive net gas exchange was immediate and linear when adipose tissue from fed and fasted, refed rats was incubated with crystalline insulin and when adipose tissue of fasted, refed rats was incubated with “bound” insulin. A delay in response was observed when adipose tissue of fasted rats was incubated with either form of insulin or when the adipose tissue of fed rats was incubated with “bound” insulin. It has been suggested that epididymal adipose tissue has the ability to modify the activity of crystalline or “bound” insulin and that it does this in response to the metabolic state of the animal.

Responses of obese-hyperglycemic mice and normal mice to 'bound' and crystalline insulin.

Abstract The effects of bovine crystalline insulin and “bound” insulin obtained from human serum on glucose utilization in intact obese-hyperglycemic mice and their nonobese siblings and on their isolated epididymal adipose tissue have been studied. “Bound” insulin showed activity similar to that of crystalline insulin in control mice. It stimulated the incorporation of glucose into diaphragm glycogen in vivo and into epididymal adipose tissue glycogen and fat in intact mice and isolated epididymal fat pads. The utilization of glucose by obese-hyperglycemic mice was depressed when compared to controls in the presence of “bound” or crystalline insulin. In older obese and control mice, their ability to synthesize epididymal adipose fat decreased. The formation of diaphragm glycogen also decreased with age in the obese but not control mice.

Effects of vitamin B6 and B1 deficiencies and restricted caloric intake on tissue zinc levels.

Studies from this laboratory have suggested that there is a decreased availability of insulin in vitamin B6 deficient rats (1). Although the role of zinc on insulin metabolism has never been determined, associations of zinc and insulin have been reported for more than 30 years. Recent in vitro studies (2,3) using isolated epididymal adipose tissue have clearly shown that zinc in concentrations in the range of those observed in human and rat plasma and higher inhibit insulin activity. Hsu (4) has reported that vitamin B6 deficiency in rats results in decreased zinc content of plasma, liver, pancreas, and heart tissues. Work in this laboratory using a different experimental protocol appeared in disagreement with Hsus conclusions. This report contains a study of the effects of vitamin B6 deficiency on tissue zinc concentrations. Since some pyridoxal enzymes are magnesium dependent and it has been shown that magnesium supplementation of vitamin B6 deficient rations for rats prevents at least in part some effects of vitamin B6 deficiency, [protects against calcium oxalate urolithiasis, increases urinary citrate and decreases xanthurenic acid excretion (5)] the effects of vitamin B6 deficiency on tissue Mg levels were also investigated. To partially determine how specific the effects observed were, studies of the effects of thiamine deficiency and restricted feeding on zinc tissue levels and the effects of restricted feeding and vitamin B6 deficiency on potassium tissue levels were also under taken. Experimental. Weanling male Charles River CD rats were used in these studies. They were fed semipurified diets containing in percent: casein, 25; sucrose, 70.7; corn oil, 9; cod liver oil, 1; salts IV (6), 4; and choline, 0.3. Eight mg of riboflavin, 40 mg of niacin, 20 mg of calcium pantothenate, 1 mg of folic acid, 1 mg of menadione, 0.2 of biotin, 0.05 mg of vitamin B12 and when used 4 mg of thiamine hydrochloride, and 4 mg of pyridoxine hydrochloride were added to each kilogram of diet. The basal diet contained 40 mg of magnesium and 1.4 mg of zinc/100 gm.

Evidence of an Insulin Inhibitor in Human and Rat Serum.

Summary Greater insulin-like activity was observed in dialyzed rat and human sera than undialyzed sera, using an isolated rat epididymal adipose tissue insulin assay technique. Dialysates from both rat and human sera decreased the activity of rat epididymal adipose tissue in the presence of crystalline insulin.

Effect of Vitamin B6 Deficiency on the Metabolism of Isolated Fat Cells: Response to Insulin, Epinephrine and Theophylline

Summary When isolated epididymal fat cells from vitamin B6-deficient rats were incubated with glucose, lipogenesis was greater both in the absence and presence of insulin than when fat cells from fed and fasted control animals were similarly treated. Fat cells from vitamin B6-deficient rats also appeared more sensitive to lipolysis induced by theophylline and to a lesser extent epinephrine. This effect appeared to be related at least in part to the inanition accompanying the vitamin deficiency. Insulin was effective in partially inhibiting the epinephrine effect on fat cells from ad libitum contols but not on fat cells from deficient rats or pair fed controls. Conversely, insulin appeared less effective in inhibiting the theophylline effect on fat cells from ad libitum controls than the other groups. Ratios of triglyceride to DNA were similar in fat cells obtained from control and deficient rats indicating that in rat vitamin B6-deficiency experiments the amount of fat cells used can be quantified by the amount of trigylceride present.