Albumin synthesis rates in patients with hypoproteinemia

812

May, I971 T h e ] o u r n a l o~ P E D I A T R I C S

All, l l l n i n syn thesis rates in patients with hypoproteinemia Albumin synthesis rates in 7 hypoproteinemic patients were determined. 75Selenomethionine, an amino acid precursor to albumin, was injected, and serial blood samples were taken over a 5 day period. Albumin was isolated from serum by Sephadex gel filtration, quantitated, and the radioactivity determined. An incorporation constant, K~,o, representing the rate of albumin incorporation of rSseIenomethionine per liter of plasma per hour was determined. The hypoproteinemic children with excessive protein loss had a mean K,,o of 9.38 • 105 c.p.m, per liter per hour compared to that of control subjects of 4.88 • lO s c.p.m, per liter per hour. Hypoproteinemic patients with severe liver disease had a mean K~,o of less than half that of control subjects (1.58 • lOS). Patients with excessive protein loss are apparently unable to adequately compensate for protein loss. They do not have a primary defect in protein synthesis.

W. Allan Walker, M.D.,* Robert A. Ulstrom, M.D., and James T. Lowman, M.D.** MINNEAPOLIS,

1V~I N N ~

loss of albumin is rapidly replenished. The production of this protein is increased to compensate for such a loss. This compensatory response is sufficient to maintain normal plasma concentration despite severe protein loss? In

I N

N O R 1VI A L

M A N

an

acute

From the Departments of Pediatrics and Radiology, University of Minnesota Medical School. Supported by Atomic Energy Commission Contract A T (1I-1) 1274 and National Institutes of Health Grant CA 08832 and the Graduate School Research Grant of the University of Minnesota. *Address: Gastrointestlnat Unit, Massachusetts General Hospital, Boston, Mass. 02114". **Present address: Department oI Pediatrics, University of Kansas Medical School, Kansas City, Ken. Vol. 78, No. 5, pp. 812-820

certain disease states, however, the plasma albumin concentration decreases in the face of continued loss into either the bowel or urine. This lower concentration suggests either an imparied hepatic synthesis of albumin or a rate of loss which exceeds the limit of compensatory ability of the liver in these diseases. 2-4 Study of albumin synthesis rates in man has usually been made by assuming a steady state in which synthesis equals degradation. The majority of these studies have utilized a preparation of albumin exogenously labeled with either iodine (131I) or chromium (51Cr) .5, 6 The difficulties associated with this method have been reviewed by Jarnum. 7

Volume 78 Number 5

Albumin synthesis in hypoproteinemia

METHODS

Table I. Patient selection

Patient

Age

F.S. 15 yr. W.W. 15 yr. J.R. 1 yr. J.G. 3 yr.

Diagnosis

Anorexia nervosa Cystic fibrosis Mental retardation Extrahepatic biliary atresia D.F. 7 yr. Anaphylactold nephrosis R.S. 2 yr. Ulcerative colitis J.B. 7 yr. Glomerulonephritis with nephrosis J.T. 2 yr. Idiopathic nephrotic syndrome G.H. 6 too. Tyrosinemia with cirrhosis S.L. 5 ~ Ino. Biliary cirrhosis D.O. 13 yr. Cysticfibrosis with cirrhosis

Serum albumin concentration*

4.6 3.6 4.4 3.4 3.2 3.1 1.5 2.4 2.7 2.3 2.5

*Serum albumin concentrations were determined by paper eleetrophoresls and are expressed in grams per cent.

Endogenous labeling of the protein with tagged amino acids such as carbon (14C) arginine, s sulfur (~5S) methionine, 9 and nitrogen (15N) glycinC ~ has produced more direct information concerning the synthesis rate of albumin. The use of these compounds in man has been limited because of the radiation dose delivered or because of difficulty in quantitating the specific tracer. The rate of incorporation of an amino acid into a specific protein directly reflects the rate of synthesis of that protein. 11, 12 In this study the rates of albumin incorporation of methionine labeled with 75 selenium (7~selenomethionine) were studied in 3 groups of patients: (1) control patients without evidence of loss of protein and with normal serum protein values, (2) patients with hypoproteinemia who had measured evidence of excessive protein loss, and (3) those with hypoproteinemia secondary to severe liver disease. The rates of incorporation of 7~selenomethionine in these 3 groups were found to be distinctly different. Patients with excessive protein loss exhibited an increased rate of incorporation, but obviously not adequate to compensate for losses. The patients with liver disease had a definitely decreased rate of amino acid incorporation.

8 13

AND MATERIALS

Patient selection. Eleven patients ranging in age from 5 months to 15 years were investigated (Table I and Appendix). A written consent was obtained from either a parent or guardian for each patient before starting the study. The patients were divided into 3 groups. Group I represents the control subjects with normal serum albumin levels and no known abnormality in albumin metabolism. Group II consisted of patients with hypoproteinemia and documented excessive loss of albumin via the bowel lumen or kidneys. Those in Group III, also hypoprotelnemic, were known to have severe intrinsic liver disease. Three microcuries of ~Sselenomethionlne (Sethotope ~) per kilogram of body weight were drawn into a syringe and diluted to 10 ml. with isotonic saline. One half of this volume was injected intravenously into each patient. Serial serum samples were obtained at 10, 30, 60, 90, 120, and 180 minutes, 6, 9, 12, and 24 hours, and then daily for 4 days. Serum protein electrophoresis was performed on each subject before and during the test period. A 24 hour quantitative urine protein determination was obtained on each patient. If excessive protein loss in the urine was found, daily quantitation of urine protein continued for the duration of the test. Patients suspected of having exudative bowel disease were tested with albumin labeled with 51chromium (Chromalbin, E. R. Squibb and Sons) 13 prior to the labeled amino acid study. The amount of ~Sselenomethionine necessary to achieve statistically significant results was calculated from animal data reported by Hine 14 and from preliminary results obtained in our laboratory. Total beta plus gamma radiation was calculated to be 0.247 tad per 1.5 ~c of 7~selenomethionine per kilogram of body weight. Albumin was separated from serum samples using gel filtration (Sephadex, Phar*E. R. Squibb and Sons (47.1 me. per milligram specific activity and 200 me. per milligram content of DL-methionine per milligram).

8 14

Walker, Ulstrom, and L o w m a n

macia Fine Chemicals, Inc., G-100) on 2.5 cm. x 45 cm. column (K 25/45) maintained at 5 ~ C. in a 0.9 per cent NaC1 and 0.02 per cent NaN8 eluant, is One-milliliter samples were applied to an ascending system with a flow rate of 12 to 15 ml. per hour. Albumin fractions repeatedly examined by both paper and immunoetectrophoresis were estimated to be greater than 95 per cent pure. To minimize nonpeptide linkage to 75selenomethionine to albumin, 0.1 ml. of 0.05N NaHSO~ was added to each sample before separation and incubated for one hour at 37 ~ C. 16 To determine the degree of contamination introduced by unbound isotope, free 75selenomethionine was incubated for one hour with human serum albumin and then placed on the Sephadex column. No radioactivity appeared in the albumin fraction. Two methods were used to quantitate the albumin faction. A spectrophotometric (280 mta) determination of the ultraviolet light absorbed by the aromatic amino acids in the albumin molecuW 7 was compared with a standard curve of known concentrations of purified bovine serum albumin (Armour Pharmaceutical Co.). This technique could not be used on icteric patients, since the absorption spectrum of bilirubin-bound albumin is altered when compared with pure albumin. The biuret method zs for determining albumin was used on these samples. The 2 methods were compared on samples from nonicterie patients and were found to yield a difference of 2 per cent or less. Albumin was expressed as milligrams per milliliter of solution. After the albumin fraction from the column was quantitated, all of the protein in that fraction was precipitated with equal volumes of 20 per cent cold trichloracetic acid, filtered on No. 1 Whatman filter paper, and washed twice with 5 per cent trichloracetic acid. The dried precipitate was counted in a sodium iodide well detector with a single channel spectrometer. The supernatant was also counted; these counts were consistently less than 5 per cent of the counts in the precipitate. Thus nearly complete recovery of

The Journal of Pediatrics May 1971

Table II. Results

Patient

K~,~~

Albumin peak

Max-

t,me

{mum

(hr.)

% total

6 3 3 3

5.0 5.0 10.8 7.3

Group I

F.S. W.W. J.R. oI.G. Mean Group II D.F. R.S. J.B. J.T.

6.40 5.27 3.26 4.60

x x • x

105 105 105 105

4.88 x 105 10.00 8.23 9.83 9.47

x x x x

105 105 105 105

Mean

9.30 x 105

Group III G.H. S.L. D.O.

1.23 • 105 1.17 x 105 2.33 x 105

Mean

1.58 x 105

1.5 1.5 2.3 3.0

10 9 6

2.1 4.7 5.6 6.2

5.8 10.0 2.0

eExpressed as the increase in albumin counts per liter of plasma per hour.

the albumin was accomplished by the precipitation. Counts were recorded to give a significant counting error of less than 2 per cent. Background counts were always less than 35 counts per minute. Calculations. The total radioactivity of the injected dose was determined as follows: The weight of the isotope injected was determined by weighing the syringe to 5 decimal places prior to and after the injection. A weighed one-gram aliquot of the remaining isotope solution was diluted 100-fold and used as a counting standard. With this standard, and appropriate calculations, an accurate percentage of the total injected radioactivity could be determined for any sample. The final results are expressed as the counts per minute contained in the albumin of a liter of plasma (counts per minute per liter). The dose of 1.5 b~c per kilogram standardized the procedure for body weight differences. This is comparable to calculating the albumin pool for a standard unit of body weight for each patient. Other methods for plotting the data such as specific activity of

Volume 78 Number 5

Albumin synthesis in hypoprotefnemfa

Group T

25

"

20

E $,

t5

LO 0 • if) C

Group TT

8 15

Group TIT

A/&-'---'-'-•AJ"8. J/ 9 el~ I /

~'x\

iO

/i/o/// / ,/// ,~~

(_) .c_

\ \~ ,A d. ~ 'd

5

E

.//

-(3

I

2

5

4

5

6

0

I

2

5

4

5

6

0

I

2

5

4

5

6

Time (hours)

Fig. 1. The rate of amino acid incorporation into albumin is demonstrated for each of the subjects in the 3 categories. The values are obtained from a weight-adjusted dose of ~selenomethionine and are expressed as the actual counts obtained from precipitated albumin when corrected for decay and calculated to represent one liter of plasma. Group I is the control group, Group II is a group of hypoproteinemic patients with excess urinary or bowel loss of protein, and Group III represents tile group with severe liver disease. This measure of albumin production is clearly different in the three groups. albumin (counts per minute per milligram), the percentage of injected dose per milligram of albumin per minut% and the percentage of injected dose in the albumin in a liter of plasma would not standardize the information to allow comparison of patients with various weights and various albumin concentrations. The count rates were plotted with time as the abscissa. The rate of incorporation for each plot was calculated by extending the early, ascending, straight line to an intercept at the 5 hour point. The rate of incorporation (Kine) was expressed as the increase in albumin counts per minute per liter per hour, i.e., Kine represents the rate of albumin incorporation of 75selenomethionine. Each value was corrected to represent an injection of exactly 1.5 /sc of 75selenomethionine per kilogram based on the exact weight of the injected isotope. RESULTS The results obtained in the 11 patients fell into 3 distinct groups (Table I I ) . Group I, the control group, had similar-appearing curves (Fig. 1). The mean of the incor-

poration c o n s t a n t s (Kine) was 4.88 x l0 s c.p.m, per liter per hour. The plateau for the incorporated, labeled, amino acid into the albumin occurred between 3 and 6 hours. The respective maximum percentages of injected dose per liter of plasma attained were 5.0 per cent, 5.0 per cent, 10.8 per cent, and 7.3 per cent. Group II, the hypoproteinemic patients with protein loss, had a rapidly ascending slope (Fig. 1). The mean of the calculated early phase slopes (Ki,,c) was 9.38 x 105 e.p.m, per liter per hour. This value is twice that seen in Group I. The time of the plateau of incorporation was between 1.5 and 3 hours. The maximum percentages of injected dose per liter of plasma were 2.1 per cent, 4.7 per cent, 5.6 per cent, and 6.2 per cent, respectively. Group t I I comprised the hypoproteinemic patients with severe liver impairment. In contrast to the other groups, the ascending slope was less steep (Fig. 1). The mean Kine was calculated to be 1.58 x 105 c.p.m, per liter per hour. The time for the plateau of amino acid incorporation ranged from 6 to 10 hours. Maximum values of injected dose per

8 16

Walker, Ulstrom, and Lowman

The ]ourna! o[ Pediatrics May 1971

methionine during this period is not accurately known, thus only limited interpretation of this data can be made. The sIope of the descending portion of the curve appears to be more steep in the control patients for the first 24 hours. A more rapid loss of the activity from the plasma of the patients in Group I I occurred in the last 96 hours.

Group I 20

.

15 i' 9j 9\

,o! i

DISCUSSION ~

_

r __

t_

1_

L__

~

__

Group I[ c~ 2 0 o~ c E 15

7~

[ ~ i ~

Ln E

g

L) c

5

m .o I

0

_&

I

__1

Group :I]I 20

15

I0

//

o~. -osc

3

6

912

2t4.

418

7~2

9'6

i2(]

Time (hours)

Fig. 2. The prolonged sampling revealed the curves for each group as demonstrated in the graphs. The significance of these findings is discussed in the text. liter of plasma were 5.8 per cent, 10.0 per cent, and 2.0 per cent, respectively. The Kin, for each group was distinctly different and there was no overlap in the individual values within the various groups. The times at which incorporation rates of 75selenomethionine into albumin attained a plateau configuration were also distinctly different from one group to the other. Fig. 2 is a graph of the values obtained for each group for the entire 120 hours of the study. T h e reutilization rate of 7~seleno

The ability of normal liver function to maintain adequate serum albumin levels despite excessive protein loss has been repeatedly demonstrated in both animal and human studies. McKee and associates, 19 by creating experimental ascites in dogs and performing daily paracenteses, were unable to lower the normal albumin concentration. Matthews, 2~ investigating the compensatory capacity of rabbits stressed with daily plasmaphoresis, noted no change in the intravascular albumin pool. In like manner, when patients with multiple myeloma were subjected to massive plasmaphoresis over long periods of time, no change in plasma albumin concentration could be detected? However, m patients with excessive protein loss due to pathologic conditions such as nephrosis or exudative bowel disease, this capacity to compensate is insufficient. Kaitz 21 documented this observation in adult nephrotie patients, and Schwartz and Jarnum, 22 as well as others, 23, 2~ have repeatedly demonstrated this in exudative enteropathies. Jarnum, T studying 8 patients with nephrosis, noted a 2- to 3-fold increase in the degradation of albumin. These same patients were either producing albumin at a normal or less than normal rate. Patients with exudative bowel disease losing one third to one half of their total vascular albumin pool per day are able to increase synthesis by no more than twice the normal rate. 22 Undoubtedly, numerous explanations for this discrepancy exist. Possibilities include a lowered steady state of albumin metabolism as part of the disease process and the lack of essential nutrients for protein production in disease states. However, one general con-

Volume 78 Number 5

sideration must be answered. Is this a diseaseassociated primary defect in albumin synthesis or simply limited compensation? To answer this question, we measured the incorporation rate of an amino acid precursor, selenomethionine, into albumin in hypoproteinemic patients with exudative loss and with severe liver disease. Traditionally, investigators have measured albumin turnover using degradation curves from exogenously labeled proteins. However, the use of labeled proteins such as ~31I-albumin has certain inherent limitations. Steady state conditions must be assumed in order to calculate synthesis rates of albumin. Under normal conditions this assumption is probably valid but in pathologic, hypoproteinemic states in which the plasma pool is constantly changing, a steady state is improbable. Schultze and Heremans 2~ feel that the formulas used to determine protein turnover under pathologic conditions are invalid. In addition, labeling techniques tend to affect the rate of degradation of the injected tracer protein? 1 Many of the classic experiments in albumin turnover utilized a high specific activity-labeling technique which may have provided inaccurate data. When a labeled amino acid precursor is injected into a patient, its rate of incorporation into a specific protein reflects the rate of production of that protein. T, ~ T o quantltate the actual rate of protein production, the intraeellular precursor pool of that amino acid and the amount of amino acid incorporated into the protein must be known3 5 Reeve and associates 8 and MaeFarlane and associates26, 27 were able to measure the rate of albumin production over a short interval of time using sodium ~4C carbonate (Na2 1~CO3) to label the guanidine carbon atom of arginine. Urea produced in the KrebsHenseleit cycle was used as an external indicator of intracellular amino acid activity. By determining the urea pool and quantitating its excretion, a direct indication of ~4C incorporation could be made and albumin synthesis could be determined. Other amino acid precursors 2s have been used to measure protein production, but because of the

Albumin synthesis in hypoproteinemia

817

problem of reutilization no specific conclusions could be drawn. Penn and associates 29 have demonstrated that with such labels as 14C and 35S reutilization becomes a significant problem. ~SSelenomethionine has certain characteristics which make it particularly well suited as an amino acid precursor. As a substitute for an essential amino acid it is rapidly incorporated into body proteins? ~ ~1 7~Selenomethionine is a gamma-emitting isotope with a physical half life of 123 days. Its characteristics as an isotope allow for relatively easy detection? 2 Unlike other precursors, the slope of protein degradation resulting from its incorporation compares favorably with that of 13~I albumin, suggesting more rapid initial incorporation and less reufilization? 2 In addition, ~Sselenomethionine albumin has a degradation curve which closely follows that of native albumin? 1 The absolute synthesis rate of albumin cannot be calculated from the data obtained in this study since the size of the native, intracellular pool of methionine cannot be measured. If some breakdown product of methionine could be isolated and quantitated, then the principles of precursor-product relationships would pertain and the absolute rate of albumin synthesis could be determined. The constants calculated from the initial ascending slope of the incorporation curve are proportional to the rate of amino acid incorporation and hence protein production. In those patients with low serum albumin levels secondary to excessive loss, the average K~nc is twice that of normal individuals. Their production rate is more rapid than the rates in normal unstressed individuals. The patients with severe intrinsic liver disease with low serum albumin values had a much lower KJne than either control patients or the hypercatabolic hypoproteinemic patients. This is presmnably due to an intracellular impairment of albumin production, i.e., a primary synthesis defect. The plateau on the curve of 75selenomethionine incorporation occurred at distinctly different times for each patient group.

8 18

Walker, Ulstrom, and Lowman

T h e early incorporation in Group I I is related to their more rapid albumin production. I n like manner, the retarded incorporation in those patients with severe liver disease implies a slow rate of synthesis. Although these patients were studied over a 5 day period, the significant time of observation was during the first 12 hours. I n this interval the Iikelihood of introducing artifact by protein breakdown and amino acid reutilization of the precursor label was markedly reduced. I n addition, variations in the albumin pool size are less significant during such a short period of observation. T h e degradation curve for each group appears to differ. T h e hypoproteinemic patients, with excessive loss of protein, had a slower early phase falloff than normal subjects, suggesting either a contracted albumin pool or slower equilibration. After 72 hours the degradation component of the curve was more steep in the hypoproteinemie patients with excessive loss, less rapid in control patients, and least rapid in the patients with severe liver disease. T h e method described demonstrates an increased production of albumin in patients with excessive loss of this protein. T h e y do not compensate completely as is evidenced by their hypoproteinemic state. T h e significantly eIevated Kinc in these patients compared to normal subjects and compared to the decreased K i ~ in h ypoproteinemic patients with liver disease would suggest a normal albumin anabolic pathway with some quantitative limitation. T h e data from the patients with severe liver disease (Group I I I ) tend to validate this method since they were shown to have predicted low values demonstrating their markedly diminished albumin production. REFERENCES

1. Adams, W. S., Blahd, W. H., Figueroa, W. G., and Bassett, S. H.: Human plasmaphoresis, Amer. J. Med. 15: 409, 1953. 2. Schwartz, M., and Jarnum, S.: Protein losing gastroenteropathy, Danish Med. Bull. 8: 1, 1961. 3. Blahd, W. tI., Fields, M., and Goldman, R.: The turnover of serum, albumin in the nephrotic syndrome as determined by I in1-

The Journal o[ Pediatrics May 1971

4.

5.

6. 7. 8.

9.

10.

11.

12.

13.

14.

15. 16.

17. 18.

labeled aIbumin, J. Lab. Clin. Med. 46:. 747, 1955. Jeejeebhoy, K. N., Samuel, A. M., Singh, B., Nadkarni, G. D., Desai, H. Q., Borkar, A. V., and Mani, L. S.: Metabolism of albumin and fibrinogen in patients with tropical sprue, Gastroenterology 56: 252, 1969. Steinfeld, J. L., Paton, R. R., Flick, A. L., Milch, R. A., Beach, F. E., and Tabern, D. L.: Distribution and degradation of human serum albumin labelled with I T M by different techniques, Ann. N. Y. Acad. Set. 70: 109, 1957. Waldmann, T. A.: Gastrointestinal protein loss demonstrated by CrhMabelled albumin, Lancet 2: 121, 1961. Jarnum, S.: Protein losing gastroenteropathy, Philadelphia, 1953, F. A. Davis Company, pp. 61-66. Reeve, E. B., Pearson, J. R., and Martz, D. C.: Plasma protein synthesis in liver: method for measurement of albumin synthesis in vivo, Science 139: 914, 1963. Volwiler, W., Goldsworthy, P. D., MacMartin, M. P., Wood, P. A., Mackay, I. R., Fremont-Smith, K., and Shook, D. F.: Turnover rates of various plasma proteins in normal and cirrhotic humans using biosynthetic methods, J. Clin. Invest. 34: 1126, 1955. Wu, H., and Bishop, C. W.: Pattern of N ~-s excretion in man following administration of N15-1abelted glycine, J. Appl. Physiol. 14: 1, 1959. McFarlane, A. S.: Metabolism of plasma proteins, in Munro, H, M., and Allison, J. B., editors: Mammalian protein metabolism, vol. 1, New York, 1964, Academic Press, Inc., p. 313. Jarnum, S.: Radioisotope techniques for the study of protein turnover, in Radioisotope techniques in the study of protein metabolism, Technical report series No. 45, Vienna, 1964, International Atomic Energy Agency, p. 94. Waldmann, T. A., and Worchner, R. D.: The use of CrS~-labelled albumin in the study of protein-losing enteropathy, in Peeters~ H., editor: Protides of the biological fluids, Proceedings of the eleventh colloquium, Amsterdam, 1964, Elsevier Publishing Company, p. 224. tIine, G. J.: Internally administered radioisotopes, in Hine, G. J., and Brownell, G. L., editors: Radiation dosimetry, New York, 1956, Academic Press, Inc., p. 866. FIodin, P., and Kil!ander, J.: Fraetionation of human serum proteins by gel filtrations, Biochim. Biophys. Acta 63: 403, 1962. Szentivany, A., Radovich, J., and Talmaje, D. W.: The separations of free disulfide-bound and peptide-bound Se75 radioactivity in serum and tissues, J. Infect. Dis. 109: 231, 1961. Wetlaafer, D. B.: Ultraviolet spectra of proteins and amino acids, Advances Protein Chem. 17: 303, 1952. Westley, J., and Lambeth, J.: Protein de-

Volume 78 Number 5

19.

20. 21. 22. 23. 24.

25.

26. 27.

28.

29. 30.

31.

32.

Albumin synthesis in hypoproteinemia

termination on the basis of copper-blnding capacity, Biochim. Biophys. Acta 40: 364, 1960. McKee, F. W., Schloerb, P. R., Schilling, J. A., Tishkoff, G. H., and Whipple, G. H.: Protein metabolism and exchange as influenced by constriction of the vena cava. Experimental ascites an internal plasmaphoresis. Sodium chloride and protein intake predominant factors, J. Exp. Med. 87: 457, 1948. Matthews, C. M. E.: Effects of plasmaphoresis on albumin pools in rabbits, J. CIin. Invest. 40: 603, 1961. Kaitz, A.: Albumin metabolism in nephrotie adults, J. Lab. Clln. Med. 53: 186, 1959. Schwartz, M., and Jarnum, S.: Gastrointestinal protein loss in idiopathic (hvpercatabolic) hypoproteinemia, Lancet 1: 327, 1959. Gordon, R. S., Jr., Bartter, F. C., and Waldmann, T. A.: Idiopathic hypoalbuminemlas, Ann. Intern. Med. 51: 553, 1959. Strober, W., Wochner, R. D., Carbone, P. P., and Waldmann, T. A.: Intestinal lymphangectasis: a protein-losing enteropathy with hypogammaglobulinemia, lymphocytoDenia and impaired homograft rejection, J. Clin. Invest. 45: 1643, 1967. Schultze, H. E., and Heremans, J. y.: Molecular biology of human proteins, vol. I, Amsterdam, 1966, Elsevier Publishing Company, p. 459. McFarlane, A. S.: Measurement of synthesis rates in liver-produced plasma proteins, Biochem. I. 89: 277, 1963. MeFarlane, A. S., Irons, L., Koj, A., and Regoeczi, E.: The measurement of synthesis rates of albumin and fibrinogen in rabbits, Biochem. J. 95: 536, 1965. Tschudy, D. P., Bacchus, H., Welssman, S., Watkin, D. M., Eubanks, M., and White, J.: Studies of the effect of dietary protein and caloric levels on the kinetics of nitrogen metabolism using N 15 labelled aspartic acid, J. Clln. Invest. 38: 892, 1959. Penn, N. W., Mandeles, S., and Anker, H. S.: On the kinetics of turnover of serum albumin, Biochlm. Biophys. Acta 26: 349, 1957. Awwad, tI. K., Potchen, E. J., Adelsteln, S. J., and Dealy, J. B., Jr.: The regional distribution of Se75 selenomethlonlne in the rat, Metabolism 15: 370, 1966. Awwad, H. K., Potchen, E. J., Adelstein, S. J., and Dealy. J. B., Jr.: Se 75 selenomethionine incorporation into human plasma proteins and erythrocytes, Metabolism 15: 626, 1966. DiGiulio, W., and Beierwaltes, W. H.: Parathyroid scanning with selenium*s labelled methionine, J. Nucl. Med. 5: 417, 1964.

APPENDIX

(CASE H I S T O R I E S )

Group I.

Patient F. S. A 15-year-old girl was admitted because of a 25 pound weight loss over a 5 month period. No evidence of chronic infection,

8 19

intestinal malabsorption, or endocrlnopathy was apparent. After a complete work-up for failure to thrive proved negative, the diagnosis of anorexia nervosa was made. Total serum proteins by paper electrophoresis were 6.6 Gm. per cent with 4.2 Gm. per cent albumin and 2.4 Gm. per cent globulins. A 24 hour urine specimen showed no protein excretion. Patient W. W. A 15-year-old boy with a miId form of cystic fibrosis was admitted for an elective surgical procedure. Liver function tests which included a percutaneous liver biopsy were within normal limits. A 24 hour urine specimen showed no protein loss. Height and weight for age were in the fifth percentile. Total serum protein was 6.6 Gm. per cent with 3.8 Gm. per cent albumin and 2.6 Gin. per cent globulins. Patient f. R. A 1-year-old boy with psychomotor retardation was admitted because of failure to thrive. Metabolic screening tests which included a urinary amino acid excretion pattern, dermatoglyphics, and a Berry spot test for mucopolysaccharides were negative. A pneumoencephalogram showed enlarged ventricles suggesting cortical atrophy. An electroencephalogram was diffusely abnormal. Liver function tests were normal. The fat content of a 4 day specimen of stool was normal. A 24 hour urine sample was normal for protein content. Total serum protein determined by paper electrophoresls was 7.2 Gm. per cent with 4.2 Gm. per cent albumin and 3.0 Gm. per cent globulin. Patient f. G. A 3-year-old boy with extrahepatic biliary atresia was admitted to evaluate dietary therapy. Liver function tests included a bilirubin level of 11.7 mg. per cent with 6.5 mg. per cent direct, a serum ornithine carbonyl transferase level of 449 units (normal, less than 25), and a liver biopsy specimen showing early portal cirrhosis. Four-day stool fat excretion while the patient was on a medium-chain fatty acid diet was 6 Gm. of fat per day (normal, 5 Gin. per day). A 5 hour D-xylose excretion was 32 per cent of the ingested dose. These tests were unchanged from tests performed 6 months earlier. Total serum protein level was 7.7 Gm. per cent with 3.4 Gin. per cent albumin and 4.3 Gm. per cent globulin. A 24 hour urine collection showed no protein content and a 5 day stool sample showed a normal excretion of CrSMabeled albumin (Chromalbin). Group II. Patient D. F. A 7-year-old girl was admitted with the diagnosis of anaphalactoid purpura and

82 0

Walker, Ulstrom, and Lowman

subacute glomerulonephritis. The hospital course was complicated by an intussusception requiring surgical intervention. The renal lesion was treated by a prolonged course of heparin and steroid therapy. During the study, the total serum protein level varied from 6.2 to 5.0 Gm. per cent, and the corresponding serum albumin levels varied from 3.4 to 2.1 Gm. per cent. Excessive protein was excreted in 24 hour urine samples (4.9 Gin. per liter of urine). The excretion of Cr51-1abeled albumin (Chromalbin) in a 5 day stool sample was 1.5 per cent of the injected dose (normal, less than 0.3 per cent). Despite extensive therapy, daily weight remained essentially constant during the study period. Patient R. S. A 3-year-old boy was admitted for evaluation of his chronic ulcerative colitis which began in the newborn period. Two years prior to this admission, a total colectomy was performed when a toxic megacolon developed. Liver function tests including a percutaneous biopsy specimen were normal. Biopsy of the ileum showed a normal mucosal pattern. Total serum protein concentration was 5.3 Gm. per cent with 2.9 Gm. per cent albumin and 2.4 Gin. per cent globulin. A 24 hour urine protein determination was normal. Excessive (3 per cent) CrSMabeled albumin (Chromalbin) was excreted into the stool over a 5 day period. Patient ]'. B. A 7-year-old boy with subacute glomerulonephritis and the nephrotic syndrome was admitted for therapeutic evaluation. Liver function tests and liver biopsy were negative. A percutaneous kidney biopsy showed proliferative glomerulonephritis. Daily urine excretion of protein varied between 4.0 Gm. per liter and 7.0 Gm. per liter. Five day Chromalbin excretion was 2 per cent of the injected dose (normal, 0.3 per cent). Total serum protein was 4.4 Gm. per cent with 1.5 Gin. per cent albumin. His weight during the period of study was stable. Patient f. T. A 2~-year-old girI with idiopathic nephrosls was admitted because of exacerbation of the disease. Liver' function tests were normal. A percutaneous kidney biopsy was interpreted to be normal. Urinary protein excretion was 1.58 Gin. per liter. No attempt was made to determine protein excretion into the intestinal lumen. Total serum protein by paper electrophoresis was 5.2 Gin. per cent with 2.4

The Journal o[ Pediatrics May 1971

Gm. per cent albumin and 2.8 Gm. per cent globulin.

Group III. Pc2ient G. H. A 6-year-old girI with tryosinemia and mild cirrhosis was admitted for evaluation of ascites. Excessive urinary excretion of tyrosine was documented by amino acid chromatography. An open liver biospy done at the time of exploratory laparotomy showed hepatocellular necrosis and regeneration. Liver function tests at the time of the study included a serum bilirubin level of 2.3 rag. per cent (1.8 mg. per cent direct bilirubin), alkaline phosphatase level of 80 units, and a prothrombin time of 15 seconds (control, 11 seconds). Total serum protein concentration was 4.3 Gin. per cent with serum albumin of 2.7 Gin. per cent and serum globulin of 1.6 Gm. per cent. Daily weights remained constant during the study period. Patient S. L. A 4-month-old boy was diagnosed as having severe extrahepatic biliary atresia and cirrhosis. He was admitted when hepatic function began to deteriorate and liver failure became imminent. Liver function tests were grossly abnormal. They included a serum bilirubin of 35 mg. per cent, a prothrombin time of 20 seconds (control, 10 seconds), and an ornithine earbonyl transferase level of 400 units. A total serum protein level determined by paper electrophoresis was 4.7 Gm. per cent with 2.3 Gm. per cent albumin. Urine excretion of protein was 1.01 Gin. per liter. Weight remained constant on diuretic therapy during the period of study. The patient developed hepatic coma and succumbed three weeks after completion of the study. Patient D. O. A 13-year-old boy with cystic fibrosis was admitted because of early hepatic failure and ascites. Liver function tests included a serum bilirnbin of 2.5 mg. per cent with 1.2 mg. per cent direct bilirubin, prothrombin time of 15.3 seconds (control, 11.0 seconds), and an ornithine carbonyl transferase level of 75 units Total serum protein was 8.8 Gm. per cent with 2.5 Gm. per cent albumin and 6.2 Gm. per cent globulin. A percutaneous liver biopsy showed marked portal fibrosis and early cirrhosis. No protein was found in a 24 hour urine collection. The ascites cleared on chlorothiazide and Aldactone therapy. During the study his weight remained unchanged.