EFFECTS OF CALORIC AND NONCALORIC MATERIALS IN FASTING HYPERBILIRUBINEMIA

Transcription

1 G ASTROENTEROLOGY 68: fi Copyright 1975 by The Williams & Wilkins Co. Vol. 68, No.2 Printed in U.S.A. EFFECTS OF CALORIC AND NONCALORIC MATERIALS IN FASTING HYPERBILIRUBINEMIA PETER V. D. BARRETT, M.D. Division of Gastroenterology, Department of M edicine, Harbor General Hospital, Torrance, and the University of California S chool of Medicine, Los Angeles, California Fasting hyperbilirubinemia (FHB) has previously been shown to be rapidly reversed by the ingestion of a mixed diet. This study examines the effect of carbohydrate, fat, amino acids, and noncaloric materials on FHB. After an initial fast of 15 to 39 hr, caloric and noncaloric materials were administered by mouth or vein to 13 subjects, and the total serum bilirubin (TSB) was determined frequently for 4.5 hr. Only oral glucose reversed FHB; the TSB did not change significantly from control studies after oral saline, mannitol, amino acids, and fat. In contrast, intravenous infusion of glucose resulted in a significant increase in TSB which could not be explained by osmotic or intravascular volume changes, and was nonspecific, since similar effects resulted from infusions of mannitol and amino acids. The results of this study demonstrate that oral glucose reverses FHB, and that under the conditions of these experiments other nutrients and noncaloric materials are ineffective. In contrast, intravenous glucose, mannitol, and amino acids increase the TSB by unknown mechanisms. Great progress has been made during the past decade in the elucidation of the biochemical pathways involved in the production and disposition of bilirubin. However, relatively little is known about the factors which modulate these pathways and affect the rate of production, clearance, and excretion of bilirubin. One of these factors is fasting, which has been shown to produce a significant increase in the concentration of serum bilirubin in normal subjects and in patients with a variety of types of liver Received October 3,1973. Accepted August 7, Address requests for reprints to: Dr. Peter V. D. Barrett, Harbor General Hospital, 1000 West Carson Street, Torrance, California This investigation was supported in part by Research Grants AM and HD 06334, and the Clinical Research Center Grant RR-425 from the National Institutes of Health. The author is indebted to Dr. Rudi Schmid for his critical review of the manuscript, to Mr. John R. Cocherell for his expert technical assistance, and to Victoria M. Adkins and Madelyn Tatum for the manuscript preparation. 361 disease. 1-4 Plasma radiobilirubin clearance studies suggest that the hyperbilirubinemia of fasting may be explained by a decreased rate of hepatic extraction of bilirubin from the plasma. 4 It has also been shown that fasting hyperbilirubinemia is rapidly reversed by feeding,3 but it is not known what aspects of feeding are responsible. This study was undertaken to examine the possible role of various nutrients administered by mouth or by vein, and of physical factors, such as osmotic and volume loads, in an attempt to identify essential factors in the reversal of fasting hyperbilirubinemia. Materials and Methods The 13 subjects involved in this investigation consisted of 4 normal subjects and 9 p atients whose diagnoses are listed in table 1. This table also lists the serum bilirubin concentration of these patients after an overnight fast, and, in some cases, for fasting periods of 39 to 48 hr. All investigations were conducted' in the Clinical Study Center.

2 362 BARRETT Vol. 68, No.2 TABLE 1. Diagnoses and bilirubin concentrations of study patients Patient Age Sex Diagnosis Serum bilirubin DirectltotalO Directltotal b mg/loomi 1 44 M Alcoholism 0.05/ / M CAR" 0.13/ M Normal 0.10/0.47 -/ M Normal 0.13/ / M Normal 0.16/ / M CAH 0.31/ / M Normal 0.12/ / F Gilbert's syndrome 0.20/ M Gilbert's syndrome 0.25/1.42 -/ F Hereditary spherocytosis 0.36/ F PBCd 1.37/ M Hereditary spherocytosis 0.29/ / F PBC 4.66/5.47 a Overnight fast (15 hr). Fast for 39 to 48 hr. c CAH, chronic aggressive hepatitis (hepatitis associated antigen-positive). d PBC, primary biliary cirrhosis. Total restriction of calories was maintained for 15 hr prior to the administration of test solutions in the majority of the studies. However, subjects with normal bilirubin concentrations receiving oral test solutions were fasted for a longer period (39 hr) in order to achieve higher bilirubin concentrations and greater statistical confidence in the determinations. In all patients, water was restricted for only 8 hr. A small needle was placed in a peripheral vein for multiple sampling. After three to four separate blood samples were obtained during a I-hr long base line period, the test material was given and blood samples were obtained for 4 to 5 hr. Oral materials were given over a 30-min period in 500 ml of water; intravenous infusions were given over a 30-min period of 1000 ml of solution. The orally administered test materials included water, saline, mannitol, glucose, amino acids (Stuart Amino Acids, Atlas Chemical Industries, Inc., Pasadena, Calif.), and medium chain triglycerides (MCT Oil, Mead Johnson Laboratories, Evansville, Ind.). Medium chain triglycerides were chosen in preference to more common types of dietary fat because the serum turbidity which followed ingestion of long chain triglycerides in'terfered with the determination of serum bilirubin. The foodstuffs were given in equicaloric amounts (400 cal). Oral saline and mannitol were given in concentrations which would provide an osmolality comparable to that of the glucose load (1220 milliosmoles). The intravenously administered test materials included saline, mannitol, glucose, and amino acids (FreAmine, McGaw Laboratories, Glendale, Calif.). The concentrations of these solutions are listed in ta bles 2 and 3. Serum osmolality determinations were performed in duplicate with an osmometer (Advanced Instruments, Newton Highlands, Mass.) and serum glucose determinations were performed by the clinical laboratory. The hematocrit was determined in triplicate on each blood sample in order to detect fluid shifts within the intravascular compartment which could influence the concentration of the total serum bilirubin (TSB). Using the mean hematocrit, corrected TSB values were calculated for each determination and compared with appropriate control solutions. The method of Jendrassik and Grof, as modified by Michaelsson et al. was used for determining the total and direct serum bilirubin concentration; each test was performed in duplicate. Previous studies in this laboratory with this method demonstrated a coefficient of variation of less than 2% for total bilirubin. 3 The mean of three or four duplicate determinations of direct or total serum bilirubin obtained during the 1 hr prior to administration of the test material was used as the control value and was considered as 100% for comparison with later determinations. To exclude the possibility that glucose or mannitol might themselves alter the diazotization reaction for bilirubin, an in vitro study was performed in which normal saline, glucose (100, 200, or 400 mg per 100 ml), or mannitol (400 mg

3 TABLE 2. Response of total serum bilirubin to oral test materials Test material Hours after administration Water control 100± ± ± ± ± ± ± ± 11 (n = 5) (102 ± 6) (106 ± 4) (105 ± 4) (105 ± 7) (111 ± 7) (117 ± 11) (117 ± 12) (116 ± 10) Glucose (n = 10) 94 ± 2 91 ± 2 c 90 ± 3 d 91 ± 3' 92 ± 3 d 95 ± 3 97 ± 5 98 ± 4 100g (97 ± 2) (93 ± 2)< (91 ± 3)e (92 ± 3) (93 ± 3)d (96 ± 3)" (99 ± 5) (98 ± 4) Amino acids (n = 3) 103 ± ± ± ± ± ± 6 119±5 121 ± 4 100g (105 ± 1) (102 ± 2) (106 ± 2) (107 ± 3) (110 ± 4) (114 ± 4) (129 ± 5) (122 ± 4) MCT' (n = 3) 105 ± 3 101±2 101±3 100 ± ± 4 98 ±5 99 ± 4 98 ± 4 48g (101 ± 3) (101 ± 3) (101 ± 4) (99 ± 4) (100 ± 3) (99 ± 5) (99 ± 3) (97 ± 3) Mannitol (n = 3) 110±4 117±4 116±2 115±3 113 ±5 118±5 120 ± ± 8 100g (107 ± 4) (110 ± 2) (110 ± 2) (107 ± 2) (107 ± 5) (111 ± 4) (114 ± 2) (108 ± 5) Saline (n = 3) 111 ± 2 110±4 98 ± 9 98 ±3 98 ± 5 103±9 93 ± ± 9 310mEq (109 ± 3) (108 ± 2) (106 ± 1) (108 ± 2) (110 ± 4) (112 ± 8) (109 ± 5) (113 ± 9) 500 ml administered in 30 min. Mean bilirubin concentration (±SEM) expressed as percentage of control value; data in parentheses represent corrections for hematocrit changes. c p < 0.01 when compared with water control. " P < 0.05 when compared with water control. e P < 0.02 when compared with water control., MCT, medium chai'll triglycerides '" 2;l &2 t-< a ::tl til CJJ CJJ t:!l t=: t>j ),: w Ol W

4 I 364 BARRETT Vol. 68, No.2 _ O _.!! I -. > C"':lC"?... O':lC'lC'lt-OOCl?<o::t!""'"'!c'oO')O')OO... C'J... r C'lC"-lC'?MC,OWWc.oOO ::.-... :: ' I. o.-i I _ O C'J1;.O NC"JCX)OOMC'lO':l... ; : :! g.-<.-<.-<.-<.-<.-<.-<.-< :::;' 1 "0 '101.- "0 ' _ I.> _III_C"I C"':) _ [ C"I... t-c()i1':)i.o... C " I cr':)(j)c'jooco mnt-c'l OO"''''... ' ' '.., 0 ' - < : : : ; ' ' - < : : : ; ' ' - <, :; I : : O ; ' > - < u _III_C'lN.-... t - C ' O l... t : l -.:rc'j >.-111'-111- o..- I' C"I... =... I' <. O c. o M M C : O t - L O b ; ; ; ; :! : o l! " : l l Q o o O ; - c ; 3 ' & ; :! I, per 100 m!) were incubated at 37 C in a Dubnoff shaking incubator with whole blood obtained from an icteric patient. Duplicate aliquots were removed before the addition of these materials, and at hourly intervals thereafter for 4 hr. The mean (±SEM) of all results, expressed as a percentage of the appropriate saline controls, C " I c ; : ; - o o r : : ( b w t : ' U? was : ; ; ± 0.5%. The results indicate that glucose and mannitol at these concentrations do : g not influence the diazo reaction and the deter 'I mination of serum bilirubin. Analyses of statistical significance were performed with Student's t-test. 6 The proportional responses of all patients with normal bilirubin concentrations, or with unconjugated h yperbilirubinemia were similar, and thus, all such subjects who received a given test material were treated as a group for purposes of statistical comparison. The responses of the 2 patients N N t : ' ; ' with N N primary biliary cirrhosis differed from the I remainder of the group, and data from these 2 = o patients' studies are presented separately. ' - < : : ' - < : : : ; ' ' ' - < - : < : : : ; ' ' - < Results Previous studies have demonstrated the presence of fasting hyperbilirubinemia (FHB) after 24 to 48 hr of fasting, but the early changes and the reproducibility of FHB have not been examined. As a basis for additional studies, a normal subject "0 '101 '101 I (subject 3) was fasted on four separate ' o occasions for periods of 39 hr, and, on one J' : ; ; occasion, 8 during a similar period of normal feeding. The results are shown graphically in figure 1 where "zero" hour represents supper at 6 PM, followed by fasting or a normal feeding pattern. A slight increase in TSB concentration may be noted during the feeding study at 15 and 39 hr at 9 AM, representing the diurnal rhythm which has previously been described During the fasting studies, the magnitude of the TSB increase was greatest between 15 and 39 hr, but even the small increase in TSB noted at 15 hr was a consistent finding. In the same normal subject, duplicate TSB determinations were performed on samples obtained at 10 PM (4 hr postcibal) and 9 AM (15 hr postcibal) of five occasions. The mean values and standard errors at 4 and 15 hr postcibal were, respectively, 0.36 ± 0.02 and 0.46 ± 0.03 mg per 100 ml (Student's paired t-test: P < 0.01). After fasting for a period of 15 to 39 hr,

5 February 1975 CALORIES AND FASTING HYPERBILIRUBINEMIA 365 the administration of water by mouth, given as a control solution, was associated with a progressive increase in TSB. After the administration of oral saline, mannitol, amino acids, or fat, the mean values of TSB, corrected for osmotic fluid shifts, were not significantly different from the control studies (fig. 2 and table 2). There was a tendency for the TSB to decrease 1.50 E QI D. CI 1.00 E z CD.75 => 0::...J.50 m => 0::.25 ILl (f) FASTED FED o HOURS FIG. 1. Fasting hyperbilirubinemia. The total serum bilirubin concentration during four fasting studies is compared to a feeding study to demonstrate the reproducibility of this phenomenon. "Zero" hours represents supper at 6 PM. after oral fat, and it is possible that a larger number of studies would have revealed a significant difference. In contrast, the administration of oral glucose produced a prompt and significant decrease in the TSB which reached a nadir at 2 to 3 hr. It is of interest that the mean decrease of 8% which occurred 3 hr after 100 g of oral glucose compares favorably with the response observed after refeeding a patient with biliary cirrhosis and a normal subject, both of whom had fasted for 44 to 48 hr. In each case, the TSB decreased approximately 7% within 3 hr after refeeding. Intravenous studies were performed with glucose and mannitol solutions of similar osmotic activity and with amino acids as a noncarbohydrate source of calories. During control studies with intravenous isotonic saline, a progressive increase in TSB was observed, which was similar to that observed during the oral control studies with water. In contrast to the effect of orally administered glucose, intravenous glucose produced a significant increase in the TSB when compared to the control studies. However, this effect is not glucose-specific, since similar changes were found after the intravenous administration of mannitol and amino acids (fig. 2 and table 3). The increased TSB level observed with each of e c: 0 u b c:.. u t z iii ::> 0:: ::::; iii ::> 0:: w Ul GLUCOSE (IV) SALINE (IV) WATER (ORAL). - q -GLUCOSE - - "? (ORAL) Te.t Material L HOURS FIG. 2. The total serum bilirubin concentration decreased after 100 g of oral glucose over 30 min, but increased after intravenous infusion of a similar quantity of glucose. The asterisks indicate those points which are significantly different from the appropriate control solutions.

6 366 BARRETT Vol. 68,No.2 these three compounds was significantly different from the TSB observed after intravenous infusions of isotonic saline. Correction of the TSB for changes in the hematocrit produced little change after the administration of isotonic solutions. Hypertonic solutions given by vein or mouth often produced parallel TSB and hematocrit changes, suggesting that shifts of fluid into, or out of the intravascular compartment had occurred. However, with the exception of the studies performed with intravenous hypertonic saline (vide infra), correction of the TSB values in these studies did not alter their statistical significance. Osmolality was determined on each sample of serum in studies performed in 7 subjects. Maximum changes were found in samples obtained 30 min after the end of the administration of each test material, and these changes were compared to the mean control value for each subject. The results, expressed as the mean milliosmolar change, with the number of studies and SEM, are given for each test material studied in this manner. The results of the oral water, glucose, and fat studies, respectively, were -1.9 (n = 4; SEM = 0.6), +3.4 (n = 6; SEM = 0.8), and (n = 2; SEM = 1.4). The results of intravenous isotonic saline, glucose, amino acids, mannitol, and hypertonic saline, respectively, were (n = 3; SEM = 0.6), +3.1 (n = 6; SEM = 1.1), +3.8 (n = 3; SEM = 0.8), +6.4 (n = 6; SEM = 0.7), and (n = 2; SEM = 0.9). No correlation was found between these results and the changes in TSB. In particular, although intravenous glucose and mannitol loads resulted in an increase in TSB, an osmotically equivalent load of hypertonic saline failed to produce an increase in the TSB, and, in fact, resulted in a decrease in the TSB when compared to the isotonic saline infusions. This decreased bilirubin concentration probably represents a dilutional change resulting from fluid shifts into the vascular space, since correction of the TSB for hematocrit changes resulted in values which approached those obtained with intravenous isotonic saline. The lack of correlation between the changes in the serum osmolarity and the TSB suggest that osmotic factors do not cause the alterations in TSB which occur after infusion of glucose, mannitol, and amino acids. Strikingly different changes in the TSB occurred after equal amounts of glucose were given by the oral or intravenous routes. In an attempt to examine possible relationships between blood glucose concentrations and changes in the TSB, glucose determinations were obtained at intervals. The control blood glucose (mean ± SEM) for seven oral and intravenous glucose studies was, respectively, 88 ± 5 and 91 ± 1 mg per 100 ml. The test solutions were given evenly over a 30- to 40-min period, and maximum blood glucose concentrations were observed 1 hr after administration had begun. These values were, respectively, 223 ± 6 and 382 ± 35 mg per 100 ml for the oral and intravenous glucose studies. Little change from control values was noted with the nonglucose test solutions. These results indicate that there is no correlation between blood glucose concentration and the changes in TSB which occurred in these studies. It should also be pointed out that the maximum blood glucose values cannot be compared to values obtained after a conventional glucose tolerance test because of the time over which the solutions were administered. The "direct" bilirubin concentration was determined in 2 patients with primary biliary cirrhosis and a stable hyperbilirubinemia. These patients were chosen because of the technical difficulties encountered in reliably measuring the low direct fraction of bilirubin in the normal subjects and patients with unconjugated hyperbilirubinemia. The results of studies in these 2 patients, shown in table 4, suggest that both the total and direct bilirubin concentrations decrease in response to an oral glucose load, in a manner similar to the response of the total bilirubin in the remainder of the subjects. However, the response of these 2 individuals to an intravenous load of glucose or mannitol seems to differ; that is, the expected increase in total bilirubin concentration failed to occur. The direct fraction of

7 February 1975 CALORIES AND FA S TING HYPERBILIRUBINEMIA 367 TABLE 4. Response of direct and total bilirubin to oral and intravenous test solutions i n 2 patients with mixed hyperbilirubinemia 2.5 Hr after administration Patient Oral studies Intravenous studies Water Glucose Saline Glucose Mannitol 11 Direct" Total Direct Total All other patients (mean Total' 106 ± 5 91 ± ± ± ± 12 ± SEM) a Quantities administered listed in tables 2 and 3. " Direct and total serum bilirubin as percentage of control hour. C Data from tables 2 and 3. bilirubin also failed to increase, but because the total bilirubin failed to increase, one cannot determine whether this response is characteristic of direct bilirubin or is related to these patients' disease process. The possibility that prolonged, essentially isotonic infusions of glucose might produce an effect on the TSB different from the effect of the acute studies described above was examined in the studies performed on 2 normal subjects and 1 patient with Gilbert's syndrome (subjects 3, 4, and 9). Duplicate serum samples were obtained from base line (zero hour) values prior to supper on the 1st day of the study, and constant infusions of either normal saline or 5% glucose in normal saline were begun immediately after supper, at a rate of 3000 ml per day for 39 hr. During this period of time the subjects fasted. At the completion of the saline control study, the mean TSB (±SEM) was 255 ± 16% of the control value, and at the completion of the glucose infusion study the mean TSB (±SEM) was 193 ± 23%. These differences were statistically significant (P < 0.05) and contrast with the results of the acute intravenous infusion studies. Discussion The results of these studies demonstrate that 100 g of glucose given orally is effective in reversing FHB, but that equicaloric amounts of amino acids and fat (medium chain triglycerides) produced no statistically significant change in the TSB concentration when compared to control studies performed with water. Saline and mannitol were also given orally as examples, respectively, of inert absorbable and nonabsorbable materials with an osmolality and volume comparable to that of the glucose load; however, no significant change in the TSB from control studies was noted after administration of these materials. A recent study by Bakken et al. 7 in the rat has shown that fasting or insulininduced hypoglycemia produces a marked increase in the activity of hepatic heme oxygenase, the enzyme system catalyzing the conversion of heme to bilirubin. The authors suggested that the enhanced heme oxygenase activity in the liver could increase hepatic heme turnover and bilirubin production sufficiently to produce the hyperbilirubinemia of fasting. Additional support for this concept was obtained by other workers who studied TSB and carbon monoxide excretion in patients undergoing subtotal fasting (400 cal daily). 8, 9 In t hese studies, the excretion of carbon monoxide, a product of heme catabolism, was utilized as an index of bilirubin production and found to increase proportionately to the TSB. The results of this study are also compatible with the thesis that FHB result!'! from increased bilirubin production, but several lines of evidence suggest that this may not be the major explanation for FHB. First, human studies by Bloomer and co-workers 4 have shown that fasting pro-

8 368 BARRETT Vol. 68, No. 2 duces a decrease in the extraction of serum bilirubin by the liver, which is of sufficient magnitude to account for the increased TSB. Second, significant increases in carbon monoxide excretion have not been found by some investigators. Bloomer et al. 4 could demonstrate only a modest increase in carbon monoxide excretion in 5 patients undergoing total caloric fasting. The reasons for conflicting observations concerning carbon monoxide excretion are not known, but one apparent difference in the experimental protocols concerns the degree of fasting, and additional work in this area is needed. Thus, although more than one factor may be responsible for FHB, current information indicates that the majority of the effect observed in man can be explained by decreased hepatic clearance of bilirubin, and the effectiveness of oral glucose in reversing FHB, as demonstrated in this study, suggests that glucose-stimulated mechanisms may enhance bilirubin extraction by the liver. The intravenous infusion of 100 g of glucose produced results which were strikingly different from the results obtained with orally administered glucose. The TSB rose promptly after the infusion and remained elevated for the duration of the 4.5-hr study. However, this effect is not specific for intravenous glucose since a similar, but more pronounced, increase in TSB was produced by the infusion of mannitol. In previous studies, Wood and Cahill,IO and, more recently, Forster et ai., 11 reported similar effects on the serum bilirubin concentration of normal subjects after the infusion of mannose, sorbitol, xylitol, and fructose. Although all of these carbohydrates share certain features, such as their small size and generally similar structure, the observation in this study that an intravenous infusion of a mixture of amino acids also increases the bilirubin concentration suggests that this effect is, indeed, a nonspecific one. Furthermore, the effectiveness of mannitol, an inert sugar alcohol, suggests that the phenomenon does not require the metabolism of these compounds. The lack of effect of infusions of hypertonic saline osmotically equivalent to the intravenous infusions of glucose and mannitol indicates that more than a simple osmotic or volume change is involved. This study cannot distinguish between increased production, mobilization of extravascular pools, or decreased hepatocyte extraction of bilirubin as a cause of the hyperbilirubinemia produced by infusion of the compounds listed above. However, based on current concepts of bilirubin metabolism in the fed and fasted states, it is likely that infusion of these compounds results in a decrease in the fractional clearance rate of bilirubin by the liver. Substantiation of this hypothesis, and resolution of questions relating to possible similarities of the bilirubin clearance defects with fasting and with intravenous infusions of various materials, await additional investigation. There are at least two possible explanations for the different effects observed after rapid administration of oral and intravenous glucose. First, a gut-related factor may be produced by oral glucose which facilitates hepatic bilirubin clearance; the intravenous effect which occurs after rapid infusion of glucose may operate by an independent nonspecific mechanism. The second possibility to be considered is a glucose-related mechanism which may facilitate hepatic bilirubin clearance after both oral and intravenous administration, but after rapid intravenous infusion this effect is partially masked by a nonspecific mechanism which acts to decrease bilirubin clearance. The latter possibility is, in fact, suggested by experiments in this study in which prolonged continuous intravenous infusions of glucose in 3 patients did result in dampening of the expected bilirubin increase with fasting. It would be of interest to know whether the direct, or conjugated fraction of serum bilirubin behaves differently from the "indirect" or unconjugated fraction under the conditions of these studies. It is known that unconjugated bilirubin represents the major component of the "total" bilirubin determination in normal subjects and in patients with unconjugated hyper-

9 February 1975 CALORIES AND FASTING HYPERBILIRUBINEMIA 369 bilirubinemia. Because of the technical difficulties encountered in reliably measuring the low direct bilirubin fraction in such inidividuals, only the total bilirubin was determined in most of the subjects in this investigation. However, the direct bilirubin is easily measured in jaundiced individuals with acquired hepatic parenchymal disease; therefore, studies were performed in 2 stable patients with primary biliary cirrhosis. In these patients, both the direct and total bilirubin concentrations decreased in response to an oral glucose load, but in contrast to all other patients studied, very little change was noted in the total bilirubin concentration after intravenous loads of glucose and mannitol. The direct fraction also showed little change. The data from this investigation do not provide a clear picture of the mechanism responsible for these observations, but one may speculate that oral glucose facilitates the clearance of both conjugated and unconjugated bilirubin, and that intravenous loads of small molecules, such as glucose and mannitol, actually block removal of bilirubin by the liver. It is possible that the excretory block present in patients with hepatic disease and hyperbilirubinemia is sufficiently severe that the additional block provided by intravenous glucose or mannitol is masked. Although it has generally been accepted that uptake and secretion of bilirubin by the hepatocyte are relatively independent steps, it is of interest that several workers have demonstrated decreased rates of bile now in animals with infusions of mannitol and galactose. Although serum bilirubin concentrations and bilirubin clearance studies were not performed in those experiments, the possible relationship between the results of the animal studies and these human studies is evident, and additional work in this area will undoubtedly be fruitful. REFERENCES 1. With TK: Bile pigments of blood, chapt 5. In Bile Pigments: Chemical, Biological, and Clinical Aspects. New York, Academic Press, 1968, p Felsher BF, Rickard D, Redeker AG: The reciprocal relation between caloric intake and the degree of hyperbilirubinemia in Gilbert's syndrome. N Engl J Med 283: , Barrett PVD: Hyperbilirubinemia of fasting. JAMA 217: , Bloomer JR, Barrett PVD, Rodkey FL, et al: Studies o n the mechanism of fasting hyperbilirubinemia. Gastroenterology 61: , Michaelsson M, Nosslin B, Sjolin S : Plasma bilirubin determination in the newborn infant. A methodological study with special reference to the influence of hemolysis. Pediatrics 35: , Snedecor GW, Cochran WG: Statistical Methods. Sixth edition. Ames, Iowa, Iowa State University Press, 1967, p Bakken AF, Thaler MM, Schmid R: Metabolic regulation of heme catabolism and bilirubin pro duction. 1. Hormonal control of hepatic heme oxygenase activity. J Clin Invest 51: , Egger G, Kutz K, Bachofen H, Preisig R: Bilirubin production in subjects with Gilbert's syndrome (GS). In The Liver. Quantitative Aspects of Structure and Function. Basel, Karger, 1973, p Lundh B, Johansson B, M ercke C, et al: Enhancement of heme catabolism by caloric restriction in man. Scand J Clin Lab Invest 30: , Wood FC, Cahill GF: Mannose utilization in man. J Clin Invest 42: , Forster H, Meyer E, Ziege M: Erhohung von Serumharnsaure und Serum-bilirubin nach hochdosierten Infusionen von Sorbit, Xylit und Fructose. Klin Wochenschr 48: , Chenderovitch J, Phocas E, Raytureau M : Effects of hypertonic solutions on bile formation. Am J Physiol 205: , Heggeness FW: Galactose and bile flow. Proc Soc Exp BioI Med 101: , 1959