Experimental Biology and Medicine

Three side chain analogs of all-rac-α-tocopherol (all-rac-α-T) were evaluated for vitamin E activity on the basis of their efficacies in preventing exudative diathesis (ED) and in supporting growth and survival of selenium-deficient chicks. Results showed that 2RS,4′RS-α-T-C₁₁ (lacking one isopentyl unit) and 2RS-α-T-nC₁₃ (with an unbranched 13-carbon side chain) had the following activities relative to those of all-rac-α-T: 69 and 50%, respectively, for supporting growth; 37 and 46%, respectively, for preventing exudative diathesis; 13 and 44%, respectively, for sustaining chick survival. 2RS-α-T-C₆ (lacking two isopentyl units) demonstrated no biological activity at the levels fed.

Intracerebroventricular (icv) injection of endogenous pyrogen (EP) into rats causes dose-related fever. To compare this procedure with the usual assay method, iv injection into rabbits, a preparation of crude rabbit EP was titrated by both methods. The rat icv injection procedure gave results similar in linearity and magnitude of response to the conventional rabbit iv method, but was found to be at least 50-fold more sensitive. The icv assay worked for human as well as rabbit EP and for partially purified as well as crude preparations. Plasma from human or animal sources did not interfere. The rat icv and rabbit iv assay methods were also compared in their ability to detect a pyrogen in filter-sterilized plasma from rats infected with the live vaccine strain (LVS) of Francisella tularensis. The rat icv method gave fevers of 1° with 0.05-ml samples of the filtered plasma from infected rats, but no fever with plasma from control rats. The pyrogen was partially purified from the infected rat plasma. It was found to be heat labile and nondialyzable, and was tentatively identified as rat EP. The less sensitive rabbit assay was unable to detect the plasma pyrogen, using 0.05- and 1.00-ml samples. These observations suggest that icv injection into rats is a useful and sensitive assay method for EP, particularly when only small volumes are available for testing.

Mixed micelles were prepared containing sodium taurocholate, monolein dioleyl lecithin, cholesterol, and an equimolar mixture of palmitic, oleic, and linoleic acids. These were incubated with commercial bile acid-sequestering resins, cholestyramine and DEAE-Sephadex, or various dietary fibers and fiber components including wheat bran, cellulose, alfalfa, lignin, and two viscosity grades of guar gum. Binding of monolein and fatty acids was determined as the difference between the radioactivity of the added micellar component, and that recovered in the centrifugal supernatant after incubation. In general, the extent of monoglyceride or fatty acid sequestration was characteristic and reproducible for each binding agent. Cholestyramine and DEAE-Sephadex essentially quantitatively bound monoglycerides and all three fatty acids from micellar medium. Low- and high-viscosity grades of guar gum sequestered 15-23% of the monolein and 32-33% of the fatty acids, showing a significant preference for linoleic acid in each case. Alfalfa fiber removed about 18% of the micellar monoglyceride and mixed fatty acids, again showing some preference for the polyunsaturated acid. Lignin, the hydrophobic component of dietary fibers, sequestered about 13% of the available lipids and displayed an apparent preference for oleic acid. Wheat bran and cellulose showed little affinity for micellar lipids binding about 11 and 4.7%, respectively. These data on resin and fiber sequestration of micellar fatty acids and monoglycerides compare favorably with the binding of other micellar components including phospholipid, bile salt, and cholesterol.

The healing rate of full-thickness skin lesions is slower in senescent female rats (22-23 months) than in their young adult (5-6 months) counterparts. Fifty percent wound closure times were 9.46 ± 0.31 and 6.45 ± 0.40 days, respectively (P < 0.001). Ovariectomy significantly slows the healing process in the mature group but not in the senescent animals. Treatment of ovariectomized rats with low-dose estradiol (0.3 μg/kg body wt) restores the healing rate to nearly normal in mature rats and accelerates the healing rate relative to sham-operated controls in senescent rats. Thus, decreased healing rates in senescent female rats may be due in part to reduced estrogenic potentiation of healing.

Embryonic chick cartilage is used to detect somatomedin activity, however, the presence of somatomedin in the developing embryo has not been reported. We have shown that thyroid hormones stimulate somatomedin activity in the rat. This study compares changes in thyroid hormones and somatomedin in the developing chick embryo. Somatomedin activity, thyroxine (T₄), and triiodothyronine (T₃) were determined in sera obtained from embryonic chicks on Days 10 through 19 of incubation and from young adult chickens. Somatomedin was determined by bioassay which measures the sulfate incorporation into pelvic cartilage of 12-day-old chick embryos. T₄ and T₃ were determined by radioimmunoassay. Somatomedin activity was not detected in 10- to 12-day-old embryos, was measurable at 13 days, and increased to a peak value at 15 days (potency of 0.93 compared to normal rat serum value of 1.0). No somatomedin was detected in the serum immediately prior to hatching or in young chicken serum. T, was not detected until Day 15 (0.42 μg T₄/100 ml) and increased to 1.39 μg T₄/100 ml on Day 19. T₃ was present in the serum throughout the study, with an increase occurring on Day 14 (51 ng T₃/100 ml). The appearance of somatomedin coincides with collagen formation and ossification in the chick embryo skeleton. The changes in thyroid hormone concentrations apparently do not influence somatomedin in the chick embryo as has been demonstrated in the rat.

The influence of the protease inhibitors, antipain and leupeptin, on type C virus induction from mouse cells by 5-iodo-2-deoxyuridine (IdUrd) and ultraviolet (uv)-irradiated herpes simplex virus (uv-HSV) was studied. Exposure of unaltered intact A1-2 cells, derived from the BALB/c mouse, to these inhibitors decreased the level of endogenous type C virus normally induced by IdUrd or uv-HSV treatment. Dose-response studies showed that antipain was more suppressive of virus induction than leupeptin. Virus induction was suppressed at inhibitor concentrations which did not appreciably affect cellular macromolecular synthesis or capacity of noninduced cells to support helper virus replication upon exogenous infection. These results suggest that the inhibitors affect the virus induction process. Our results are similar to those reported for antipain inhibition of λ-prophage induction in Es-cherichia coli, and corroborate a recent report on suppression of mammalian virus induction by these protease inhibitors.

These studies were designed to determine the presence of an angiotensin-converting enzyme in turkeys. Captopril, a converting enzyme inhibitor, prevented the elevation of diastolic, systolic, and mean blood pressures which occurs characteristically in response to administration of angiotensin I. However, the responsiveness of blood pressure to administration of angiotensin II and norepinephrine was maintained. Thus, the results suggest the presence of an angiotensin-converting enzyme in turkeys.

The content of zinc, magnesium, calcium, bilirubin, and bile acids was determined in the hepatic bile of zinc-deficient and control swine. Zinc deficiency was produced by dietary zinc restriction while pair fed zinc supplemented animals served as controls for observations of hepatobiliary functions. Pigs weighing 5 kg were placed in two groups and fed the respective diets for 6 weeks; hair and skin abnormalities as well as decreased weight gain were present in the zinc-deficient group by this time. Hepatic bile and pancreatic juice were obtained from each animal after careful isolation and cannulation of the pancreatic and bile ducts. Animals were studied for a 3-hr period while under constant secretin stimulation and chloralose anesthesia. During this period, there was a progressive decrease in the biliary concentrations of bilirubin, bile acids, and magnesium in the bile of both groups, while zinc and calcium levels were not altered. The zinc concentrations in pancreatic juice were reduced in the zinc-deficient animals. The zinc content of the serum, liver, kidney, and pancreas was also decreased in this group. The calcium and magnesium content of the serum and organs was similar in the two groups with the exception of decreased magnesium in the kidney of the zinc-deficient pigs. Low content of zinc in the liver and serum of the deficient animals was associated with a zinc concentration in the bile comparable to the control group; however, the relative content of zinc in pancreatic secretion and bile was found to be altered in zinc deficiency. In control animals 60% of the zinc is in the pancreatic secretion and 40% in the bile. These percentages are reversed in zinc-deficient animals in which 60% of the zinc is derived from the bile.

A radioimmunoassay (RIA) for cytosolic glycerol-3-phosphate dehydrogenase (GPDH) was developed as a means of determining enzyme concentration independent of assayable activity. The GPDH concentration (nmole/g wet wt), activity (units/g wet wt), specific activity (units/nmole GPDH), total liver concentration (nmole/liver/kg body wt), and total liver activity (units/liver/kg body wt) were determined under conditions favorable and unfavorable to gluconeogenesis. Fasted rabbits showed a decrease in total liver nanomoles of GPDH per killigram of body weight. Cortisone, triamcinolone, and thyroxine administration caused increases in total liver units and total liver nanomoles of GPDH per killigram of body weight in the fed animal. The increases or decrease observed correlated directly with changes in liver size. Diabetic, insulin-controlled diabetic, and glucagon-treated fed animals showed neither GPDH activity nor concentration changes. There was no change in the specific activity of GPDH under any conditions. The constant specific activity observed indicates a single catalytic state for the enzyme. Thus, changes in total liver enzyme rather than in specific activity become critical in the regulation of the GPDH reaction.

R-Type serum vitamin B₁₂-binding proteins (cobalophilins) are a heterogenous population of glycoproteins. The reason for this appears to be due to variation in the carbohydrate moieties of the macromolecules. To support this contention we have used different sugar-specific lectin-sepharose affinity columns as a means for separating cobalophilins. Concanavalin A-Sepharose can resolve serum vitamin B₁₂-binding activity into three distinct fractions. The first two are cobalophilins and the third is transcobalamin II. The major cobalophilin fraction does not interact with concanavalin A-Sepharose, but is bound completely by both wheat germ and castor bean II lectins. Elution from these columns can be accomplished by addition of the appropriate competitive sugar to the buffer. Castor Bean I Lectin-Sepharose resolves the major concanavalin A-Sepharose cobalophilin fraction into two distinct fractions—one which does not interact with this lectin and the other which is eluted when N-acetylgalactosamine is added to the buffer. Furthermore, the distribution of exogenous-bound (⁵⁷Co) cyancobalamin between these two fractions is different when comparing certain myeloproliferative diseases in which serum cobalophilins are usually elevated. It is suggested that use of lectin—Sepharose resins may be a more definitive method for defining cobalophilin variants than is presently available.

The relationship between renin and renal prostaglandin E₂ (PGE₂) release was investigated in anesthetized dogs using a highly specific radioimmunoassay for PGE₂ measurement. There was a dissociation between the acute inhibition of renin release with intra-renal infusion of angiotension II (AII) or angiotensin III (AIII) and renal PGE, release. Intrarenal infusion of norepinephrine resulted in a significant increase both in renin and renal PGE₂ release. Intrarenal infusion of bradykinin increased renal PGE₂ but not renin release, and infusion of isoproterenol increased renin release but not renal PGE₂ release. Renal pressure reduction from 134 to 71 mm Hg increased renin release while the renal PGE₂ release was unaltered. In addition, renal vasoconstriction was observed with an infusion of norepinephrine, AII, and AIII, while renal vasodilation was observed with an infusion of isoproterenol and bradykinin, and renal pressure reduction. Neither renal vasoconstriction nor vasodilation, however, correlated with renal PGE₂ release. These results suggest that the renin-angiotensin system probably does not play a regulatory role in the control of renal PGE₂ release, and that the change of renal vascular tone does not accompany renal PGE₂ release.

Using isolated intestinal segments in rats, the absorption of iron, lead, and cobalt was increased in iron deficiency and decreased in iron loading. Similarly, the absorption of these metals was decreased in transfusional erythrocytosis, after intravenous iron injection and after parenteral endotoxin injection. Acute bleeding or abbreviated intervals of dietary iron deprivation resulted in increased iron absorption from isolated intestinal segments and in intact animals, while the absorption of lead and cobalt was unaffected. These results suggest that the specificity of the mucosal metal absorptive mechanism is either selectively enhanced for iron absorption by phlebotomy or brief periods of dietary iron deprivation, or that two or more mucosal pathways for iron absorption may exist.

A radioisotopic assay for the cytoplasmic deoxycorticosterone sulfotransferase activity (DSA) of rat liver was developed herein. Deoxycorticosterone inhibits the enzyme reaction strongly. For reliable, quantitative results, a complex assay method, using several different deoxycorticosterone concentrations, each studied with four different amounts of enzyme, was necessary. This “mosaic” assay compensated for variations of the enzyme activity observed in male and female rats. The results obtained demonstrated that cytosols from female rats contained five to six times the DSA found in males. The hepatic DSA was examined further by DEAE-Sephadex A-50 fractionation. One peak of enzyme activity was observed in cytosols from males and two peaks of enzyme activity were observed in cytosols from females. Comparison of these DSA peaks with the glucocorticoid sulfotransferases (STI, STII, and STIII) already fractionated in this laboratory, suggested that the single peak of DSA in males and the second peak of DSA eluting from DEAE-Sephadex A-50 columns in females were STIII. The first peak of DSA eluting from the ion-exchange columns in females appeared to be a new enzyme.

Prostacyclin (PGI₂) is readily converted to 6-keto-prostaglandin F_1α (6-keto-PGF_1α) which is both a stable and biologically less potent compound. Recently, another stable metabolite of PGI₂ namely 6-keto-prostaglandin E₁ (6-keto-PGE₁) was found to be equipotent to PGI₂ in inhibiting platelet aggregation and reducing renal vascular resistance. In this study, cardiac, pulmonary, and systemic vascular responses to exogenously administered 6-keto-PGE₁ were characterized and compared with responses to PGI₂ Systemic arterial diastolic pressure (DP), pulmonary arterial pressure (PAP), and myocardial contractile force (MCF) were measured in intact dogs following intravenous (iv) and intraarterial (ia) administration of PGI₂ (0.1, 0.3, 1.0 μg/kg), 6-keto-PGE₁ (0.1, 0.3, 1.0 μg/kg), prostaglandin E₂ (0.3, 1.0, 3.0 μg/kg), and 6-keto-PGF_1α (1.0, 3.0, 10.0, 30.0 μg/kg). 6-Keto-PGE₁ was found to be equipotent to PGI₂ in reducing DP following either iv or ia administration. 6-Keto-PGE₁ mimics the response to PGI₂ in reducing PAP and MCF when both are administered intravenously. All responses to PGI₂ and 6-keto-PGE₁ are dose dependent. 6-Keto-PGE₁, like PGI₂, is unaffected by pulmonary transit. These results suggest that vascular responses previously attributed to PGI₂ may be due in part to 6-keto-PGE₁.

Coxsackie B3 virus produces myofiber necrosis and mononuclear infiltration in the hearts of both BALB/c and athymic mice within 8 days after infection. Viral clearance time from the myocardium of both athymic and BALB/c mice did not differ. Within 2 weeks of infection both strains of mice develop chronic inflammatory infiltrates comprised of cells that were diffusely and intensely acid-α-naphthyl esterase positive and characteristic of monocytes or showed only focal esterase staining characteristic of T-lymphocyte lineage.

The relationship between the reproductive system and calcitonin secretion was investigated in the rat. Plasma levels of calcitonin were measured in normal and ovariec-tomized rats at basal levels and during calcium challenge. Sensitivity of the normal and operated animals to exogenous calcitonin administration was also studied. Ovariectomy leads to a transient fall in both CT levels and in the secretion of the hormone in response to a calcium challenge. However, a greater sensitivity of ovariectomized animals to salmon calcitonin is maintained after the return to normal of both the basal level of the hormone and of its secretion in response to a calcium challenge. Estrogen substitutive therapy does not restore CT levels and antagonizes the action of exogenously administered CT in ovariectomized rats. These results suggest the existence of a direct or indirect effect of ovarian factors, other than estrogens, on CT secretion.

The effects of selected pharmacological agents on mucin secretion by the tracheal epithelium of piglets were studied using organ culture. Mucin release into the culture medium was reduced, and mucin retention by secretory cells of the mucosa and submucosal glands was increased, by colchicine, vinblastine sulfate, and cytochalasin B. Dibutyryl cyclic AMP had no significant effect. Although these agents have diverse and poorly understood influences on cell functions, the results suggest a possible role for mi-crotubules and microfilaments in the intracellular movement and release of mucin.

A drug-resistant subpopulation of cells (HTFU) was previously isolated from the human colonic carcinoma cell line HT29. The cell lines varied significantly in in vitro growth patterns with HTFU cells showing contact inhibition while HT29 cells demonstrated uncontrolled growth (10). The malignant cell lines were grown in the presence of various concentrations of blood group A fucolipid, Lewis_b, Forssman hapten, and ceramide tri-hexoside for 2 weeks. Although HTFU cells demonstrated an equal or enhanced sensitivity to the lipids relative to HT29 cells, the cell lines responded in similar fashion to each lipid. Treatment with Forssman and ceramide trihexoside induced a temporary inhibition of cell proliferation, however, continued exposure to these lipids became stimulatory. Exposure to Lewis_b and blood group A fucolipids caused a permanent retardation of cell proliferation.

A substance recovered from the medium of embryonic chick bones growing in organ culture inhibits the proliferation of chick calvarial bone cells in vitro. This inhibition of proliferation occurs within 3 hr after exposure of the cells to the inhibitory substance (IS) in a manner which does not appear to affect the protein synthesis or the gross morphology of the cells. The medium from which IS is obtained can be diluted 500-fold and still retain significant inhibitory activity. Cells in culture spontaneously escape the inhibition of a single dose of IS within 24 hr of exposure, however, upon further addition of IS to these cells inhibition of proliferation is again observed. Cells derived from chick bone as well as rat and human bone are more sensitive to the inhibition than are chick dermal fibroblasts and chick liver and muscle cells. IS is not destroyed after treatment of the conditioned medium with trypsin, phospholipase A₂, RNase, neuraminidase, or heat. IS is also not a commonly produced prostaglandin. Estimates of the molecular weight of IS range from 6000 to 14,000 daltons. While these features do not disclose the exact nature of this substance, they are consistent with this substance being a small polypeptide without the exposed amino acids, lysine or arginine. We conclude that the presence of such a substance supports the concept of local regulation of bone metabolism.

Interest in the relationship of uric acid (UA) to insulin stems from reports of a possible association between gout and development of glucose intolerance as well as the structural similarity between uric acid and the diabetogen, alloxan. Previous attempts to examine this relationship using experimental animals possessing a potent uricase have produced inconsistent results. In order to examine the effects of elevated UA concentrations on serum-immunoreactive insulin (IRI), lactic acid, and glucose, we used rats treated with potassium oxonate (KOx), a competitive inhibitor of uricase, to prevent rapid degradation of UA. Male Wistar rats fed 3.5% KOx or 3.5% KOx plus 2% UA in a modified AIN-76 diet for 1 week had serum UA and whole blood lactic acid increased twofold. Hyperuricemic rats drank more water and weighed less than controls. In the 1-week experiments we describe here, animals treated with KOx alone (endogenous UA) did not become hypoinsulinemic compared to controls whereas those fed KOx and UA (endogenous plus exogenous UA) showed a decrease in serum IRI of 44%. In longer-term experiments (4 weeks), it was sufficient to feed 4.5% KOx alone to observe a 26% decrease in insulin. Thus, it appeared that the effect was dose and time dependent. Serum glucose increased (24-38%) in animals fed KOx/UA for 1 week. As a result, insu1in:glucose ratios also decreased in the KOx/UA-fed group suggesting abnormal insulin secretion. Results from in vitro studies using isolated neonatal rat pancreatic islets indicated that UA, unlike KOx, rapidly suppressed insulin secretion by up to 65%. The rapidity of the suppression and subsequent reversal of the effect by removing UA from the medium further suggested that UA interferes with insulin secretion. These data indicate a reexamination is warranted of the possible cytostatic if not cytotoxic effects of U A toward the β-cells of the pancreas.

The possibility that circadian phase of NaCl intake may play a role in the development of hypertension was evaluated with the use of DOCA-implanted rats, receiving either saline (1%) or water as follows: Group I, saline ad libitum; Group II, saline from 6 PM to 6 AM; Group III, saline from 6 AM to 6 PM; Group IV, water ad libitum. All animals were kept on a 12:12 light schedule (lights on from 6 AM to 6 PM) and regular rat chow ad libitum. The mean daily fluid intake (ml/100 g body wt/24 hr) during the first 2 weeks of the experiment was: 57 ± 1.5; 59 ± 1; 45 ± 1; and 25 ± 1.6 for Groups I, II, III, and IV, respectively. On the third experimental week, the mean systolic BP (mm Hg) ± SE was as follows: Group I, 154 ± 4.1; Group II, 140 ± 3; Group III, 154 ± 5; Group IV, 132 ± 4.5. The data suggest that hypertension develops more readily among rats ingesting excessive salt loads while at rest (circadian phase of low natriuresis) than among those doing so during the active phase (circadian phase of high natriuresis) of their day.

Models for producing toxic liver injury and cirrhosis have been developed in rats and applied to the study of drug pharmacokinetics, but lack of a well-characterized model of hepatic congestion has prevented previous investigation of the effects of elevated hepatic venous pressure on drug metabolism in the rat. Chronic hepatic congestion was produced in rats by partial ligation of the inferior vena cava immediately below the diaphragm and above the entrance of the hepatic veins. Significant elevation of hepatic venous pressure, prolongation of prothrombin time, and pericentral fibrosis, and sinusoidal dilitation on histological examination were seen 6 weeks after initial surgery. Liver weight in the hepatic congestion group was significantly increased when compared to sham-operated controls, while no significant difference was seen between the two groups with regard to body weight gain, bile flow, bile acid output, serum bilirubin, and alanine aminotransferase. Pentobarbital pharmacokinetic parameters were determined after intravenous administration of [¹⁴C]pentobarbital to animals with hepatic congestion and sham-operated control animals. Plasma disappearance of pentobarbital was prolonged in rats with hepatic congestion and systemic plasma clearance was reduced. Additional studies of drug metabolism in this rat model of hepatic congestion may suggest important questions for future clinical investigation in man.

Previous studies indicate that cardiac hypertrophy is associated with altered mechanical properties including prolonged contraction times and mechanical refractory periods and exaggerated aftercontractions in isolated rat papillary muscle preparations. The present study was designed to ascertain whether changes in transmembrane potentials accompany the altered mechanical properties. Responses of papillary muscles from hypertrophied hearts of deoxycorticosterone acetate-treated Wistar Kyoto (WKY) rats were compared to those of nonhypertrophied, sham-treated WKYs. Muscles connected to a force transducer in a bathing chamber containing a 27° oxygenated physiological salt solution were subjected to field stimulation and isometric tension measured. Intracellular microelectrodes were used to measure transmembrane potentials of single impaled cells. When compared to nonhypertrophied controls, the hypertrophied preparations had (1) longer action potentials and contraction times, (2) longer electrical and mechanical refractory periods, and (3) exaggerated transient depolarizations and aftercontractions following paired-pulse or high-frequency stimulation. Propranolol given to block the effect of endogenous catecholamine released by field stimulation decreased the electrical excitability and the tension output of all preparations but did not eliminate the differences between the hypertrophied and nonhypertrophied groups.

Summary. Characteristics of simultaneously recorded membrane potentials and isometric tension responses of isolated papillary muscles from hypertrophied and nonhypertrophied rat hearts were determined. When compared with nonhypertrophied preparations, hypertrophied preparations were found to have longer action potentials, longer contraction times, and longer electrical and mechanical refractory periods. When subjected to paired-pulse or high-frequency, short-duration stimulation, hypertrophied preparations developed larger aftercontractions and larger transient depolarizations than did nonhypertrophied preparations. Propranolol treatment of the preparations did not eliminate these differences between hypertrophied and nonhypertrophied preparations, suggesting that endogenous catecholamines were not responsible for the differences.

The effects of a biologically active metabolite of PGI₂, 6-keto-PGE₁, were evaluated in fetal goats and newborn lambs using an in situ, constant-flow, isolated lower left lobe preparation. Intrapulmonary injections of 6-keto-PGE₁ (0.074-4.41 μg/kg) produced dose-dependent decreases in pulmonary vascular resistance, mean systemic arterial pressure, and heart rate in fetal goats. Fetal systemic responses to 6-keto-PGE₁ were significantly less following left atrial injections than after intrapulmonary injections. Newborn lambs also responded to intrapulmonary infusions of 6-keto-PGE₁ (0.078-5.15 μg/kg-min) with dose-dependent reductions of pulmonary vascular resistance and systemic arterial pressure. A possible role for 6-keto-PGE₁ in the modulation of perinatal pulmonary vascular resistance is discussed.

The metabolism of copper by cultured fibroblasts from brindled male (Mo ^br/Y) and normal male (+/Y) mice was studied using radioactive copper (⁶⁴Cu). Brindled fibroblasts accumulated more ⁶⁴Cu than normal fibroblasts. The accumulation of ⁶⁴Cu by both brindled and normal fibroblasts was enhanced if the cells were preincubated in a copper-supplemented medium or if fetal calf serum was omitted from the medium. Brindled fibroblasts also retained more ⁶⁴Cu on continued incubation. The greater accumulation might, therefore, represent an effect of reduced copper efflux. The demonstrated expression of the inherited defect in copper metabolism of brindled mice in cultured fibroblasts emphasizes the value of this model for further study of copper metabolism and its genetic control.