Altered bile composition during cholesterol gallstone formation: cause or effect?

Gallbladder stasis, increased gallbladder absorption, and elevated biliary levels of calcium, hydrogen ion, and bilirubin have been implicated as factors potentially critical to cholesterol crystal precipitation. Previous studies, however, have analyzed bile only when crystals or gallstones have already formed. Therefore, we tested the hypothesis that changes in bile composition are a late effect, occurring only after crystal formation. Adult male prairie dogs were fed a standard nonlithogenic control diet (n = 7) or a lithogenic 1.2% cholesterol diet for 5, 9, or 14 days to cause cholesterol saturation (n = 7), cholesterol monohydrate crystals (n = 7), or gallstones (n = 7). Gallbladder bile was examined microscopically for crystals, and analyzed for ionized calcium, bilirubin, pH, total protein, and biliary lipids. The ratio of gallbladder to hepatic bile radiolabeled cholic acid specific activity (Rsa) was calculated as an index of gallbladder stasis. Cholesterol saturation index was calculated. The results demonstrate that increased gallbladder bile cholesterol saturation and total protein concentration precede cholesterol monohydrate crystal precipitation. However, changes in gallbladder ionized calcium, unconjugated bilirubin, pH, stasis, and absorption were noted only after crystals and gallstones had already formed. These data indicate that alterations in gallbladder bile calcium, bilirubin, pH, stasis, and absorption are not early changes, but occur simultaneously with or after crystal formation. Increased biliary protein, however, which was elevated prior to nucleation, may be an important mediator of cholesterol precipitation in cholesterol-saturated bile.     

Pigment gallstone formation and altered ion transport

Feeding corn and alfalfa to young prairie dogs resulted in formation of gallstones composed of 45 percent cholesterol, 30 percent bile pigments, and 25 percent calcium bilirubinate in half of the animals. This diet also resulted in increased gallbladder bile concentrations of calcium, phospholipids, and cholesterol. Although the cholesterol saturation index was significantly increased compared with control subjects, it remained less than 1. In addition to the changes in biliary lipid composition, the corn and alfalfa-fed animals had significantly decreased transepithelial potential difference and short-circuit current when compared with control animals. These are changes in gallbladder mucosal function similar to those that have been reported in humans with gallstones. This model may therefore prove to be of great value in studying the pathogenesis of noncholesterol gallstones.     

Effects of resection or bypass of the distal ileum on the lithogenicity of bile

Changes in the composition and lithogenicity of gallbladder bile after resection and bypass of the distal ileum were investigated in the prairie dog. In animals fed a trace cholesterol diet, both ileal resection and ileal bypass increased the cholesterol saturation of bile. In animals fed a cholesterol-enriched diet, the cholesterol saturation was increased by ileal resection but not by ileal bypass. In the animals fed the trace cholesterol diet, both ileal resection and ileal bypass induced the formation of bilirubinate gallstones.     

Bile composition and bile acid pool size. Comparison after truncal, selective, and highly selective vagotomy

We studied biliary lipid composition and bile acid pool size in 29 patients surgically treated for duodenal ulcer. Fourteen were examined both before and after surgery, the rest postsurgically only. They were divided into three groups according to type of vagotomy. With duodenal fluid obtained via nasogastric tube, we determined bile acid pool size, bile concentrations, and lithogenic index. We found no significant differences in bile composition and bile acid pool size among the three types of vagotomy, postsurgically. However, patients studied before surgery, compared with the entire post-vagotomy group, had a significant increase in relative cholesterol content and lithogenic index, most pronounced in the truncal vagotomy group. Bile acid pool size was also increased postsurgically. Vagotomy may predispose to gallstone development by increasing the bile's relative cholesterol concentration and thus the lithogenic index. However, the slightly expanded bile acid pool size may improve cholesterol solubility in certain patients.     

Cholesterol crystals in biliary microlithiasis

A recent study put forward the hypothesis that microlithiasis may represent an early stage in the development of biliary calculi. It is an established fact that cholesterol crystals are the product of an inevitable stage in the sequence leading to gallstone formation. To test the hypothesis stated above ten patients affected by gallbladder cholesterol microlithiasis (CM) were examined in the lipid composition of the bile, the cholesterol saturation index and the presence of cholesterol crystals being calculated. The results were compared with those of 14 patients affected by pigment microliths, 24 with larger stones (LS) and ten control patients. The cholesterol saturation index was above one in all CM patients, whereas in some LS patients the gall-bladder bile was not supersaturated. Cholesterol crystals were observed in the gallbladders of all CM patients and seven LS patients. These results would seem to provide support for the hypothesis of microcalculi as being "young stones", with the bile of CM patients maintaining the conditions leading to gallstone formation.     

Increased biliary cholesterol secretion in alloxan diabetic mice

Plasma and liver cholesterol levels and biliary cholesterol, phospholipid and bile acid concentrations were examined in normal and alloxan diabetic mice fed ordinary and 0.5 per cent cholesterol diets. The plasma and liver cholesterol levels markedly increased in the diabetic mice, and the cholesterol diet further increased the liver cholesterol level but not that in the plasma. The gallbladder bile weight increased in the diabetic mice, but not after the cholesterol diet. The biliary lipid concentrations markedly increased in the diabetic mice, and the increases of the cholesterol and phospholipids exceeded that of the bile acids, resulting in increases of the cholesterol molar concentration ratio (mole percent) and the lithogenic index. The cholesterol diet increased the biliary cholesterol concentration and slightly the phospholipid, but not the bile acids. Therefore, the cholesterol mole percent and the lithogenic index increased. Among the biliary bile acid composition, cholic and deoxycholic acids increased and beta-muricholic acid decreased in the diabetic mice, whereas the cholesterol diet feeding decreased cholic acid and increased chenodeoxycholic and alpha-muricholic acids. These data suggest that the mechanism of the increase of biliary cholesterol secretion in diabetic mice is different from that after cholesterol diet.     

Increased biliary cholesterol secretion in alloxan diabetic mice

Plasma and liver cholesterol levels and biliary cholesterol, phospholipid and bile acid concentrations were examined in normal and alloxan diabetic mice fed ordinary and 0.5 per cent cholesterol diets. The plasma and liver cholesterol levels markedly increased in the diabetic mice, and the cholesterol diet further increased the liver cholesterol level but not that in the plasma. The gallbladder bile weight increased in the diabetic mice, but not after the cholesterol diet. The biliary lipid concentrations markedly increased in the diabetic mice, and the increases of the cholesterol and phospholipids exceeded that of the bile acids, resulting in increases of the cholesterol molar concentration ratio (mole percent) and the lithogenic index. The cholesterol diet increased the biliary cholesterol concentration and slightly the phospholipid, but not the bile acids. Therefore, the cholesterol mole percent and the lithogenic index increased. Among the biliary bile acid composition, cholic and deoxycholic acids increased and β-muricholic acid decreased in the diabetic mice, whereas the cholesterol diet feeding decreased cholic acid and increased chenodeoxycholic and α-muricholic acids. These data suggest that the mechanism of the increase of biliary cholesterol secretion in diabetic mice is different from that after cholesterol diet.     

Ileal resection-induced gallstones: altered bilirubin or cholesterol metabolism?

Ileal resection has been shown to increase the risk of cholelithiasis. Earlier studies in humans suggested that ileal resection increases the cholesterol saturation index. Recent data from patients on long-term parenteral nutrition and from animals, however, have suggested that ileal resection predisposes to pigment gallstone formation. We therefore tested the hypothesis that ileal resection alters bile calcium and bilirubin metabolism without affecting the cholesterol saturation index. Adult male prairie dogs underwent either sham laparotomy (eight prairie dogs) or ileal resection (16 prairie dogs). All animals were fed a trace cholesterol (nonlithogenic) diet before and for 4 weeks after operation. Pigment gallstones were present in 44% of the ileal-resected animals and in none of the sham animals (p less than 0.05). Calcium bilirubinate crystals were present in 94% of the ileal-resected animals and in none of the sham animals (p less than 0.01). Gallbladder bile calcium (25.6 +/- 2.4 versus 17.2 +/- 1.1 mg/dl; p less than 0.05) and total bilirubin (29.3 +/- 4.0 versus 9.4 +/- 1.8 mg/dl; p less than 0.01) concentrations were significantly greater in ileal-resected animals. The cholesterol saturation index of gallbladder bile, however, was no different in ileal-resected (0.53 +/- 0.04) and in sham-operated animals (0.50 +/- 0.04). Although initial studies suggested that the cholesterol saturation index of hepatic bile was increased after ileal resection, a second set of experiments demonstrated that this phenomenon resulted from washout of bile salts that were already in extremely low concentrations in hepatic bile. We conclude that alterations in bilirubin, but not cholesterol, metabolism result in pigment gallstone formation after ileal resection.     

Effect of dietary ethanol on gallbladder absorption and cholesterol gallstone formation in the prairie dog

Dietary ethanol has been reported to protect against cholesterol gallstone formation. Because enhanced gallbladder absorption of water is important in cholesterol cholelithiasis, we examined the hypothesis that ethanol acts by inhibiting the absorptive function of the gallbladder. Eighteen adult male prairie dogs were fed a lithogenic liquid diet containing 0.4% cholesterol. Half of the animals received 30% of total calories as ethanol, whereas their pair-fed controls received equicaloric amounts of maltose-dextrin. After 3 months, the gallbladders were inspected for gallstones and crystals, and gallbladder and hepatic bile were analyzed. Cholesterol stones and crystals were present in all nine controls. None of the alcohol-fed animals had stones, but four had cholesterol crystals. Gallbladder cholesterol, phospholipids, and total calcium were significantly decreased in alcohol-fed animals. In both gallbladder and hepatic bile, the cholesterol saturation index was significantly lower in alcohol-fed animals, as was the ratio of trihydroxy to dihydroxy bile salts. The ethanol-supplemented diet produced a significant decrease in the absorption of water by the gallbladder as indicated by changes in the gallbladder bile to hepatic bile ratios of the total bile salt concentration (7.29 +/- 1.25 versus 3.84 +/- 0.56; p less than 0.05) and the total calcium (3.37 +/- 0.24 versus 2.43 +/- 0.29; p less than 0.05). These findings indicate that the protective effect of ethanol may be related to its ability both to inhibit gallbladder absorption of water and to alter the composition of biliary lipids.     

The effect of jejunoileal bypass on bile composition and the formation of billiary calculi

In our series of 101 patients with small bowel bypass for morbid obesity, nine developed biliary calculi postoperatively during a mean follow-up of 29.6 months. The development of gallstones depends in part on biliary cholesterol saturation and on the zeta potential of bile. In eight consecutive patients, the lithogenicity of bile was assessed by the methods of Small, Swell and Isaksson, which are dependent on cholesterol saturation. Postoperatively, the lithogenic score decreased in six and increased in two patients, one of which developed gallstones. Taurine bile salt conjugation tends to prevent aggregation of micelles by increasing the zeta potential. The biliary glycine/taurine ratio increased (p less than 0.05) from 4.6 to 5.9 postoperatively. These results suggest that the increased incidence of cholelithiasis following small bowel bypass is not only due to a relative change in bile composition but is probably more significantly due to an increase in the biliary glycine/taurine ratio and a consequent decrease in the biliary zeta potential.     

Bilirubin monoglucuronide promotes cholesterol gallstone formation

Recent evidence suggests that cholesterol (Ch) solubility in bile is determined by a complex interaction of mixed micelles and lecithin-cholesterol vesicles. Bilirubin monoglucuronide (BMG), which binds to bile salts and incorporates into mixed micelles, may displace cholesterol from micelles into vesicles, thus favoring cholesterol monohydrate crystal precipitation. Therefore, we designed an experiment to test the hypothesis that BMG may enhance cholesterol gallstone formation without inducing cholesterol supersaturation. For 8 weeks, 28 adult male prairie dogs were fed either a control, nonlithogenic diet (0.03% Ch), a high carbohydrate diet (CHO) which has no cholesterol but increases hepatic bilirubin secretion, or the same CHO diet plus 0.03% Ch. Cholecystectomy was then performed, and bile was examined microscopically for stones or crystals and analyzed for BMG and biliary lipids. Cholesterol saturation index was calculated. Cholesterol gallstones were found in none of the control animals and in 13% of the CHO-fed animals. However, the addition of trace cholesterol to the CHO diet resulted in an 88% incidence of cholesterol gallstones (P less than 0.001 vs control, P less than 0.01 vs CHO, respectively). Gallbladder bile was unsaturated with cholesterol in all groups. (control = 0.65 +/- 0.05, CHO = 0.46 +/- 0.05, CHO + 0.03% Ch = 0.70 +/- 0.03). CHO feeding alone or with trace cholesterol significantly elevated gallbladder bilirubin monoglucuronide, phospholipid, and cholesterol concentrations when compared to controls. These data suggest that in the prairie dog a high carbohydrate diet with only trace amounts of cholesterol increases bilirubin monoglucuronide in gallbladder bile and causes cholesterol gallstone formation without inducing cholesterol supersaturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leptin-resistant obese mice do not form biliary crystals on a high cholesterol diet

INTRODUCTION: Human obesity is associated with leptin resistance and cholesterol gallstone formation. Previously, we demonstrated that leptin-resistant (Lep(db)) obese mice fed a low cholesterol diet have enlarged gallbladders, but a decreased cholesterol saturation index, despite elevated serum cholesterol. Obese humans, however, consume a high cholesterol diet. Therefore, we hypothesized that on a high cholesterol diet, leptin-resistant mice would have cholesterol saturated bile and would form biliary crystals. METHODS: Eight-week old female lean control (n = 70) and leptin-resistant (n = 72) mice were fed a 1% cholesterol diet for 4 weeks. All animals then had cholecystectomies. Bile was collected, grouped into pools to determine cholesterol saturation index (CSI), and examined for cholesterol crystals. Serum cholesterol and leptin were also measured. RESULTS: Gallbladder volumes for Lep(db) mice were enlarged compared with the lean mice (35.8 microl versus 19.1 microl, P < 0.001), but the CSI for the Lep(db) mice was lower than for the lean animals (0.91 versus 1.15, P < 0.03). The obese animals did not form cholesterol crystals, whereas the lean animals averaged 2.2 crystals per high-powered field (hpf) (P < 0.001). Serum cholesterol and leptin were also elevated (P < 0.001) in the obese animals. CONCLUSIONS: These data suggest that Lep(db) obese mice fed a high cholesterol diet have increased gallbladder volume and decreased biliary cholesterol saturation and crystal formation despite elevated serum cholesterol compared with lean control mice. We conclude that the link among obesity, diet, and gallstone formation may not require hypersecretion of biliary cholesterol and may be related to the effects of diabetes, hyperlipidemia, or both on gallbladder motility.     

Caffeine prevents cholesterol gallstone formation

Methylxanthines are known to inhibit in vitro gallbladder absorption. Increased gallbladder absorption has been observed during formation of cholesterol gallstones. Therefore we tested the hypothesis that caffeine would inhibit in vivo gallbladder absorption and thus prevent formation of cholesterol gallstones. Sixteen adult male prairie dogs received a control nonlithogenic diet, and 16 were fed a diet containing 1.2% cholesterol. Half of the animals in each group received caffeine in their drinking water. Gallbladder and hepatic bile were examined microscopically and analyzed for biliary lipids and electrolytes. The gallbladder/hepatic bile ratios of bile acids and sodium were calculated as indices of gallbladder absorption. All eight animals receiving the 1.2% cholesterol diet formed cholesterol gallstones, whereas none of the eight animals fed the cholesterol diet plus caffeine formed gallstones. The cholesterol saturation index was similar, however, in both groups. In animals fed a control diet, the administration of caffeine significantly increased hepatic bile flow and decreased the gallbladder/hepatic bile ratio for both bile acids (5.4 +/- 0.9 vs 3.6 +/- 0.3; p less than 0.05) and sodium (1.26 +/- 0.03 vs 1.12 +/- 0.03; p less than 0.01). In animals fed the high-cholesterol diet, caffeine significantly decreased the ratios for both bile acids (9.0 +/- 1.6 vs 5.3 +/- 0.6; p less than 0.05) and sodium (1.37 +/- 0.06 vs 1.21 +/- 0.01; p less than 0.05), lowered gallbladder bile protein levels, normalized gallbladder stasis, and lowered serum cholesterol levels. In summary, caffeine prevented formation of cholesterol gallstones in this experimental model. The effect of caffeine may be the result of alterations in multiple biliary parameters including the inhibition of gallbladder absorption.     

Segmental adenomyomatosis of the gallbladder predisposes to cholecystolithiasis

Background/Purpose The aim of the present study was to clarify the association between adenomyomatosis of the gallbladder and cholecystolithiasis.Methods A cholecystectomy was performed for cholelithiasis or various other conditions in 1099 patients, of whom 608 had cholecystolithiasis. Adenomyomatosis of the gallbladder was classified as one of three variants: segmental, fundal, and diffuse. Segmental adenomyomatosis has an annular stricture dividing the gallbladder lumen into the neck compartment and the fundal compartment. Bile lipid analysis was performed in 8 patients with segmental adenomyomatosis.Results Adenomyomatosis of the gallbladder was observed in 156 patients (14.2%), of whom 99 had segmental adenomyomatosis, 54 had fundal adenomyomatosis, and 3 had diffuse adenomyomatosis. The prevalence of cholecystolithiasis was higher in patients with segmental adenomyomatosis (88.9%) than in those without adenomyomatosis (52.3%; P < 0.001). Gallstones were detected earlier in patients with segmental adenomyomatosis than in those without (P < 0.001) and were located predominantly in the fundal compartment. Bile in the fundal compartment had lower concentrations of total bile acids (P = 0.012), with an increased cholesterol saturation index (P = 0.012), compared to bile in the neck compartment.Conclusions Segmental adenomyomatosis is a condition predisposing to cholecystolithiasis, probably due to the lithogenic environment in the fundal compartment. Fundal or diffuse adenomyomatosis appears to be unrelated to cholecystolithiasis.     

The possibility of prediction of cholesterol saturation of gallbladder bile

Using stepwise regression we studied the possibility to determine the cholesterol saturation index (CSI) of gallbladder bile from analysis of fasting serum bile acid, 24 hour urine bile acid or serum lipids (cholesterol and triglyceride) in 31 gallstone patients. Two multiple regression equations were obtained to predict the CSI of bile from fasting serum bile acid compositions based on absolute concentration and relative concentration. The CSI of the gallbladder bile could not be predicted from 24 hour bile acid or serum lipids. Four critical values were calculated from regression equations, which predicted the CSI of bile with the efficiency above 70 percent. This study indicated for the first time that the prediction of CSI of gallbladder bile can be obtained from an analysis of fasting serum bile acid and hence it is possible to prevent gallstone formation at the stage of cholesterol supersaturated bile production.     

Bile studies after liver transplantation.

An analysis of bile composition following orthotopic liver transplantation in the rhesus monkey showed that during rejection only small quantities of viscid bile were produced and that this was associated with increased cholesterol saturation. Bile composition in patients after liver transplantation also showed that bile was supersaturated with cholesterol in the early postoperative period while the enterohepatic circulation of bile acids was interrupted by a draining T tube. However, further study of patients showed a poor correlation between bile composition and the development of biliary complications. An analysis of bile 'sludge' showed that after transplantation two types were encountered. The first, containing large quantities of unconjugated bilirubin, was present when intrabiliary obstruction was associated with long-standing mechanical obstruction. The second type, present in patients developing masses of intrabiliary 'sludging' shortly after transplantation, consisted mainly of necrotic donor biliary tract due to damage during preservation. An intrabiliary perfusion technique was developed which in animal models reduced the extent of donor biliary damage.     

Alcohol protects against cholesterol gallstone formation.

Epidemiologic studies have suggested that alcohol intake may protect against cholelithiasis. Gallstone formation was studied in 20 prairie dogs fed a 0.4% cholesterol-supplemented liquid diet. In ten animals, ethanol provided 35% of total calories. In ten pair-fed controls, ethanol was replaced with isocaloric maltose. After 3 months the gallbladders were inspected for gallstones, and gallbladder bile was analyzed. Cholesterol macroaggregates were present in all controls and pigment concretions were noted in five. No stones were observed in ethanol-fed animals. Bile in the ethanol group contained less cholesterol than the controls (5.60 +/- 0.71 vs. 9.16 +/- 0.61 mmol/L, p less than 0.05) while phospholipids, total bile acids, and bilirubin were unchanged. The resulting cholesterol saturation index was reduced in the ethanol group (0.81 vs. 1.22, p less than 0.05). The ratios of trihydroxy to dihydroxy bile acids were also different (2.07 +/- 0.25 in ETOH vs. 3.29 in controls, p less than 0.05). The bile calcium concentration was higher in control animals presumably secondary to the use of complex sugars (5.36 +/- 0.37 vs. 3.77 +/- 0.32 mmol/L, p less than 0.05). These results confirm that ethanol inhibits cholesterol gallstone formation. They further suggest that this effect is dependent on reductions of biliary cholesterol and selective changes in bile acid concentrations.     

单纯胆囊结石患者胆囊胆汁淀粉酶改变及其意义探讨

目的探讨单纯胆囊结石患者胆囊胆汁淀粉酶(amylase,AMY)的改变及其意义。方法影像学诊断明确的单纯胆囊结石患者50例,均行LC术,术中取胆囊胆汁3 mL,采用干化学法检测AMY,与血AMY比较,并对切除的胆囊行常规病理检查。结果 50例患者中胆囊胆汁AMY为30~3 020 U/L,平均(474.7±753.2)U/L,与血清AMY(64.3±17.1)U/L比,胆汁AMY明显升高(P0.05)。其中胆固醇性结石19例,胆色素性结石14例,混合性结石17例,胆色素性结石组和混合性结石组均高于胆固醇性结石组的胆汁AMY(P0.05)。腺肌症胆汁AMY水平高于非腺肌症的AMY水平(P0.01)。结论胆囊结石患者存在胰胆反流,胆汁中AMY升高可能与结石形成及胆囊的病理学改变密切相关。     

PDZK1敲除对小鼠肝脏胆固醇代谢和胆囊结石形成影响的实验研究

目的探讨敲除PDZK1(Postsynaptic density-95,disks-large,ZO-1-domain K1,PDZK1)基因对小鼠肝脏胆固醇代谢调节和胆囊结石形成的影响。方法雄性成年PDZK1基因敲除小鼠(PDZK1 knockout,KO组)和野生型小鼠(wild type,WT组),每组各10只,以成石饲料分别喂养4周,观察胆囊成石情况,并收集肝脏和胆囊组织。采用蛋白印迹法测定肝脏PDZK1和清道夫受体B族1型(scavenger receptor B type 1,SRB1)表达。采用胆总管插管收集肝胆汁,测定胆汁分泌率和胆汁胆固醇含量。采用试剂盒酶法测定胆囊胆汁成分并计算胆汁胆固醇饱和指数(cholesterol saturation index,CSI)。以实时定量PCR检测肝脏脂质代谢相关基因的mRNA表达。结果成石饲料喂养4周后,WT组小鼠全部成石(10/10),KO组小鼠则为40%(4/10)成石。两组小鼠肝胆汁分泌率差异无统计学意义,但KO组小鼠肝胆汁胆固醇含量显著降低(P0.05),胆汁酸含量增加(P0.05),且CSI降低(P0.05)。KO组小鼠肝脏SRB1蛋白表达降低(P0.05),甾醇氧-酰基转移酶基因1/2mRNA表达降低(P0.05),而肝型脂肪酸结合蛋白1和胆汁酸转运相关蛋白(ATP结合盒b11)表达则显著增加(P0.05)。结论 PDZK1影响SRB1在小鼠肝脏中表达,降低对高密度脂蛋白胆固醇摄取,减少胆汁胆固醇分泌,继而降低胆囊结石形成。     

A special, supplemented 'vegan' diet for nephrotic patients.

High dietary protein intake, in the past recommended for nephrotic syndrome, does not improve hypoproteinemia and may accelerate progressive renal damage. In contrast, low-protein diets reduce proteinuria and preserve renal function in experimental renal models of nephrotic syndrome. In this study, 20 steroid-resistant, nephrotic patients were treated with a pure vegetarian, low-protein diet, supplemented with essential amino acids and ketoanalogues (supplemented vegan diet, SVD) for 4.6 +/- 3.1 months. Before the study, these patients followed an unrestricted protein, low-sodium diet (LSD). Proteinuria, daily urea nitrogen excretion and creatinine clearance decreased significantly on SVD. A similar lowering effect of SVD was observed on serum total cholesterol. Seven of the 20 patients changed from LSD to SVD and vice-versa on 3 occasions, and in all cases, we found an increase of proteinuria during the LSD period. Serum albumin, HDL cholesterol, triglycerides and anthropometric measurements did not change on SVD. Our data suggest that SVD exerts a favorable effect on proteinuria and hypercholesterolemia in nephrotic patients, without inducing clinical or laboratory signs of malnutrition.