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An Unusual Form of Macroamylasemia
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C'opHi ~ ht
©
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()7: J49- Jf>4. 1974 ll\' Tht· \l'illi"n" 8; \l'ilk ins (',_
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CASE REPORTS AN UNUSUAL FORM OF MACROAMYLASEMIA TEHRIEt. BEHGGRE:\ A:\ D
M ICI-IAF.t. D.
L Ev rrr
Depa rtment of MPdicin P. University of Minnesota Hospitals. Minneapolis. M innesota
The apparent molecula r weight of previously st udied macroa mylases is considerably larger than norma l se rum amylase, and th e existence of macroamyl ase has therefore been easi ly docum ented by crude gel filtration t ec hniques. This report desc rib es a patient with a persistantly elevated serum amylase level resulting from an unusual macroamylase which was only marginally larger than normaL The slightly increased size of this enzyme markedly inf1uenced its ren al clearance, and the existence of this macroa mylase was first suspected because of the patient 's consistently low C .\m: C cr ratio. Doc umentat ion of the increased size of this enzyme required careful gel filtrat ion study. Isoelectric focusing showed this macroamylase to have a lower isoelectric point than did the two major components of normal serum amylase. Th e develop m ent of mild pancreatitis after pan creatic duct cannulation provided a uniqu e opportunity to observe the serum and urin e a mylase values that occur when pancreatitis is superimposed on an underlyin g macroa mylase mi a . The existence of an abnormally large form of se rum amylase ("macroamylase") has been demonst rat ed in approxim ately 45 patients. t-t' Although apparently not injurious to health , m ac roamylase is nevertheless clinically important in that it usually causes hyperamylase mia, and failure to recogni ze this entity may result in an erroneo us di agnosis of pancreat ic disease . Di agnos is of this condition has been relatively simple since the large size of the macroamylase (gre ater than 100,000 mol wt in all cases) precludes its renal clea rance, and , as a result , the hyperamylasemia has been consistantly associated with a normal or low urinary a mylase. The Rece ived October 5, 1973 . Acc epted January 10. 1974. Address requests for repr ints to: Dr. Michael D. Le vi tt, Box 346, Un iversity of Minnesota . Depart me nt of M ed icin e, Medical Sc hool, Mayo Memorial Bu ildin g, Minn ea polis, Minn esot a 55455 . Supported by Nat ional Institut es of Health Grant 5 R01 AM1 3309. 149
large size differential between m acroamylase and normal amylase (mol wt of 55,000' 2 ) also makes it possible to easily document the presence of the large enzyme by simple gel filtration screening techniques. 13 . t • This report describes serial studies of serum and urinary amylase in a patient with a heretofore undesc ribed form of macroamy lase which was only fractionally larger than normal. The correct diagnosis was initially missed because she frequently had a sli ghtly elevated urin ary amylase, and the screening gel filtration test for macroamylase was negative. However, a persistently low renal clearance ratio of amylase to creatinine (C Am: C cr) suggested the presenc e of an abnorma ll y large amylase 15 which was then documented by more refined gel filtration studies. This case demonstrates the value of the simple meas urement of C Am : C cr in assessing hypera mylasemia. Of additional interest is the fact that the patient developed
CASE REPORTS
150
mild pancreatitis after pancreatic duct cannulation, thus providing a unique opportunity to study the changes in serum and urinary amylase which occur when pancreatitis is superimposed on an underlyin g macroamylasemia.
Case Report The patient, a 69-year-old housewife, was referred to the University of Minnesota Hos pitals for evaluation of abdominal pain associated with persistent hypera mylasemia, ra ngin g from 164 to 1800 Somogyi units during the 18- mon t h period prior to admission. For the previous 5 years, she ha d ha d one to three attacks eac h yea r of dull epigastric pa in often associated with vo miting . These attacks lasted 4 to 8 hr, a nd then cleared without specifi c therapy. She denied diarrhea, fever, weight loss, ja undi ce, or a pprec iable ethanol in gest ion. Because of easy fatiguability. she had spent much of the past 2 yea rs in bed. Physical exa mina t ion was entirely within normal limits . There was no evidence of muscl e wea kn ess , an d the easy fatiguability was thought to be of functional origin. Laboratory studies which were within normal limits includ ed: hemoglobin , white blood count , erythrocy te sed imen tat ion ra t e, urinal ys is, serum creat inine (0.9 per 100 ml) and creatinine clearance (104 ml per 1.73 m 2 ), se rum electrolytes. calcium. phosphorous. carotene, cholesterol. trigl~·cericle s, bilirubin. SGOT, a lka line phosphatase. a nd electrophores is . A glucose tol era nce test was no rmal, as was a n exercise electroca rdi ogram . A 4-dav stool collection showed 1.9 g. of fat per clay: Serum lipase was normal on two occasions. A rapid sc reenin g test for mac roa mylase usin g Sephadex G-100, as described by Friel ha ndler and Berk , 13 was negati\·e on two occas ions in that no a my lase activ ity was obsen·ed in th e fract ions conta inin g blue dextran. Chest X -ray a nd a fl at plate of th e abdo men were normal. An upper gastroint est in a l seri es re\·ea led a deformed duodenal bulb without e\·idence of a n act i\·e ulcer. The small bowel follO\\·-throu gh was normal. as was a barium enema. Endoscopy re\·ea led a normal sto mac h a nd duo de num . a nd cannulation of th e s phin cte r of Odcli with retrograde injection of contrast mat eri a l re\·ea led com pl etely norm a l pancrea ti c an d bilian· ducts and a normal ga llbladder. During th~ inj ec tion oft he contrast material. the patie nt complained of se\·ere epigast ric pa in. which. along with assoc iat ed hypotension. cleared in abo ut 5 min. Aft er cannula-
Vol . 67, No.1
tion s he had a mild epigastric pa in , an elevated temperature ranging from 99 to 101 F and her previously norma l whit e blood count increased to 11 ,250 with 84o/r neutrophils. These symptoms were interpreted as being indicati\·e of mild pancreatitis , and th ey clea red after several d ays of n asogastric suction.
Studies of Serum and Urinary Amylase M ethods . Serum and urinary a my lase were assayed by a sacc harogenic technique using 3, 5-dinitrosalizylic acid as a reagent. On e unit of a mylase ac ti vity is defined as that a ctivit y which liberates 1 mg of maltose per hr at 37 C. One of these units per 1 ml is roughly comparable to 13 Somogyi units, and the upper limits of norm a l (mean + 2 SD) for serum a nd urinary a mylase (2-hr urine collection) a re 11.4 U per ml and 48 U per min , res pecti vely. 1 5 The ratio of CAm: Cc, was calculated using standard clearance formulae . 1 6 The serum sample for these calculations was obtained approximately at the midpoint of the 2-hr urine collection. The lower and upper limits of norma l are 1.1 and 4.1 %, respectively. 1 5 The apparent molecular size of amylase was determined by gel filtration of 1-ml samples on 2.5- by 40-cm columns packed with polyacrylamide (B io-Gel P-150) or Sephadex G-100 . Except when noted columns were equilibrated with and eluted with 0.02 M phosphat e buffered saline (pH 7 .0). To det ermine if the macroamylase dissociated at an acid pH , the sa mpl e was dialyzed against 0.02 M glycine-HCl buffer (pH 3.4) , and then was chromatographed on a polyacrylamide column equilibrated with this acid buffer. Blue dextran and cytochrome c were added to the sa mpl e to be chromatographed to serve , res pectively, as intern a l m ar kers of the void volum e and elution volume of a substance with molecular weight of 13,000, respectively. In order to compare and quantitate objectively the elution patt ern of a mylase from one column run to the next , the percentage of the total amylase activity eluting prior to the peak of s imultan eo us ly chromatographed cytochrome c (measured at optical density 400) was det ermin ed . Isoelectric fo cus ing was ca rried out on a 110-ml column using pH :3.5-10 carrier amph olyte (LKB Instruments. Stockholm , Sweden). Th e density gra di ent consisted of 0 to 50'i sucrose with a final a mphol yte concentration of Fi. The se rum to be focused was dialvzed against distilled water, a nd 1 ml of the s~rum was then applied to the column. Focusin g was carried out at 4 C for 48 hr . Approximately 60
CASE REPORTS
July 1974
fractions were coll ected , and each fraction was analyzed for amylase, pH, and protein (optical density 280).
Results Serial measurements of serum amylase , urinary amylase, and C Am: C cr are graphically shown in figure 1. The serum amylase was persistently elevated, but it fluctuated from the upper limits of norm al to 9 times normal. The only episode of abdominal pain during this hospitalization occurred on October 11 and 12 after duct cannulation . Thus, there was no relation between abdominal symptoms and the serum amylase level. Urinary amylase ranged from high normal to slightly greater than normal , with the exception of the two greatly elevated values obtained after the duct cannulation on September 11, 1972. With the exception of the two postcannulation values, the CAm: C cr was always below normal levels. The apparent development of pancreatitis after cannulation resulted in a dramatic rise of C.:.. m: C cr to supranormal levels. Isoelectric focusing (fig . 2) of five serum samples from normal subjects showed that serum amylase normally has two major components with isoelectric points of ap-
151
proximately 5.3 and 5.9, respectively . Three of the serum samples had a small shoulder at a pH of about 4.8 on the pH 5.3 peak (see fi g. 2). In contrast , the bulk of the patient's serum amylase had an isoel ectrical point of about 4.7 with minor peaks at 5.3 and 5.8 (fig. 2B) . Gel filtration patterns on polyacryla mide of amylase activity of normal serum , a typical macroamylase serum, and the patient's serum are shown in figure 3. The peak concentration of albumin , blue dextran , and cytochrome c are indicated by arrows . Normal serum amylase (fig. 3A) elutes slightly faster than cytochrome c in a relatively symmetrical peak. Typical macroamylase serum (fig. 3B) has an amylase component which elutes in the void volume . As shown in fi gure 3C, the patient's serum amylase peak was asymmetrical , and a larger proportion of the patient's serum amylase appeared to elute prior to cytochrome c than was the case with normal serum amylase, suggesting that a portion of her amylase was larger than normal. In order to determine objectively whether this was a reproducible finding , five different samples of the patient's serum and four different normal sera were chromatographed and t he frac-
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152
CASE REPORTS
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FRACTION FIG . 2 . Isoel ect ri c foc usin g of th e amylase of a ty pical norm al serum (A) a nd th e patient ·s serum (B ) . Th e isoelectric points of t he a mylase peaks are indi cat ed. Norm al serum has two major co mponent s wi th isoelectric points of approxim ately 5.3 and 5.9. T he bulk of t he pati ent 's serum amy las e has an isoelect ric point of roughly 4.7 , with two small peaks a t the isoelectric points of normal serum amylase .
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Fract ion FIG . 3. Gel filtr a tion elut ion pa ttern on polyacryla mide of amylase from: A , normal se rum; B, ty pical mac roamylase serum ; a nd C. the se rum of the patient described in this pa per. Th e peak concentrations of blue dextran , albumin , a nd cy tochrome c are shown by arrows.
tion of the total amylase activity eluting ahead of the cytochrome c peak was calculated. A mean of 81 ± 3.4% (1 SEM) of the patient's serum amylase eluted before the
Vol. 6 7, N o. I
cytochrome cas compared with 62 ± 2.1% of normal serum amylase, a significant (P < 0.01) difference. The difference in elution patterns between normal serum amylase and the patient's serum amylase was also observed when gel filtration was carried out on Sephadex G-100. Gel filtration may markedly underestimate the quantity and perhaps the apparent size of the macroamylase complex since some complexes dissociate during gel filtration at neutral pH. 9 · 1 0 Long and Kowlessar 10 showed that increasing the volume of the sample to be chromatographed will tend to reduce this dissociation. The elution pattern of 5 ml of the patient's serum was virtually identical to that of 1 ml , suggesting that the pattern obtained with the 1-ml sample did not represent a much larger macroamylase which was rapidly dissociating during chromatography. When gel filtration was carried out at pH 3.4, the patient's serum amylase and normal amylase eluted in identical fashion , suggesting that the patient's macroamylase represented an acid dissociable complex. In an attempt to ascertain whether amylase was bound by a dialyzable material, a sample of serum was exhaustively dialyzed against pH 3.4 buffer . The pH of the sample was then raised to pH 7.0 with NaOH, incubated at 37 C for 1 hr, and gel filtered at pH 7 .0. The elution pattern of amylase was identical to that of the patient 's untreated serum (82% of amylase eluted prior to cytochrome c peak) indicating that the complex remained intact and that the binding factor had not passed into the dialysate . Several studies have suggested that salivary amylase is cleared more slowly by the kidney than is pancreatic amylase . 17 • ' 8 To determine whether the patient's serum amylase might represent her salivary amylase, saliva was diluted 1 :50 and chromatographed. The elution pattern was identical to that of normal serum amylase with 61 % eluting prior to the cytochrome c peak. Discussion This case further broadens the spectrum of the already heterogenous condition
July 1974
CASE REPORTS
known as macroamylasemia. The abnormal enzyme has been found in patients with a variety of disease states as well as normal subjects identified by routine screening. 14 As was the situation in this case, abdominal pain has been a common accompaniment ofmacroamylasemia. This relation may well be fortuitous, however, in that abdominal pain leads to measurement of serum amylase activity, thus uncovering an unrelated macroamylasemia. Although the cause of the abdominal pain in our patient was not elucidated, the normal pancreatic duct study makes it quite unlikely that she suffered from chronic pancreatitis. Berk et al. 19 have recently described 3 patients with an elevated serum amylase level, low renal CAm: C cr. and no obvious macroamylase in the serum. Ion exchange chromatography showed that the bulk of the serum amylase of these patients behaved like one of the two major amylase components of normal serum which they have termed "salivary type" amylase. In contrast, it is clear from isoelectric focusing studies that the amylase of our patient was not one of the major components of normal serum amylase. In addition, the possibility that her serum amylase was primarily salivary amylase was also excluded by gel filtration studies which showed that her serum amylase eluted more rapidly than did salivary amylase. The apparent molecular weights of previously studied macroamylases have ranged from 160,000 to greater than two million. 2 ' 3 Although the dissociation of macroamylase complexes makes it difficult to be certain ot molecular size, the macroamylase of our patient appeared to be marginally greater than that of normal amylase. Demonstration of this small size differential required careful comparison of the elution patterns of amylase and cytochrome c, and it is possible that similar cases have gone undetected. The minimally increased size of the patient's amylase did, however, reduce its rate of -clearance by the kidney, resulting in a consistently low renal CAm :Cc, (except for the values obtained after the pancreatic duct study). The renal excretion of amylase appears
153
to result from glomerular filtration without appreciable tubular reabsorption. 17 The dimensions of the glomerular "pore" are such that for molecules in the size range of amylase, minor differences in molecular radii result in large differences in glomerular permeability. For example, egg albumin, amylase , and serum albumin which have respective molecular weights of 45,000, 55,000, and 68,000, are filtered at rates of 22%, 20 2.5%, 15 and less than 0.1 %21 of the glomerular filtration rate. The permeability of the glomerulus to amylase is most readily expressed as the ratio of CAm: Cc,, a value which normalizes for individual differences in glomerular filtration rate. 15 ' 16 The finding of a low CAm: Cc, in a patient with ' an elevated serum amylase level usually suggests the presence of macroamylasemia since this ratio is consistently elevated to supranormal levels in acute pancreatitis. 15 One explanation for this increased clearance in pancreatitis is that pancreatic amylase is more rapidly cleared by the kidney than is salivary 17 • 18 or endogenous serum amylase. 17 As shown in figure 1, a dramatic increase in CAm: Ccr occurred when mild pancreatitis (after pancreatic duct cannulation) was superimposed on the preexisting macroamylasemia. Presumably in this situation, the amylase binding substance was completely saturated, and the free pancreatic amylase was rapidly cleared by the kidney. However, the possibility that pancreatitis decreased the binding of amylase could not be ruled out. The hallmark of macroamylasemia is a normal or low urinary amylase in the face of an elevated serum amylase. It is apparent from figure 1, however, that very high serum amylase levels in the absence of pancreatitis were associated with mildly elevated rates of urinary amylase excretion . This finding probably reflects the relatively small size of our patient's macroamylase, which made possible a slow but appreciable rate of glomerular filtration whereas the glomerulus is totally impermeable to the larger forms of macroamylase. The high serum amylase levels in macroamylasemia are thought to be the result of slow metabolic clearance of the abnor-
154
CASE REPORTS
mal enzyme, rather than excessive deposition of amylase into the blood, as is the case in pancreatitis. The question thus arises as to how our patient could excrete excess quantities of amylase in her urine , if, presumably, amylase was entering her serum at a normal rate. One explanation is that studies in baboons have shown that, of the total metabolic clearance of serum amylase, only about 20% is removed in the urine while 80% is cleared by extrarenal mechanisms. 17 If the slightly increased size of the patient 's amylase hindered extrarenal clearance to a greater extent than renal clearance, the urinary amylase could be elevated even though normal quantities of amylase were entering the serum. The elution pattern of the patient's serum amylase was indistinguishable from that of normal serum amylase after treatment with an acid pH, suggesting that her macroamylase represented an acid dissociable complex of normal-sized amylase bound to an abnormal substance. These amylase-binding substances have been shown to be IgA in 2 cases, 2 IgG in 1 case, 7 and apparently something other than immunoglobulin in the bulk of the cases. The slight difference in size between the macroamylase in this case and normal amylase indicate that the binding substance was smaller than the immunoglobulins. Presumably, the serum amylase level in macroamylasemia is largely determined by the amount of binding substance in the serum. The wide swings of serum amylase levels observed in our patient over a period of several days differs from most previous cases of macroamylasemia, and suggests that there were rapid fluctuations in the serum concentration of this amylase-binding substance. REFERENCES 1. Wilding P, Cooke WT, Nicholson GI: Globulin-
bound amylase: cause of persistently elevated levels in serum. Ann Intern Med 60:1053-1059, 1964 2. Ueda M, Berk JE, Fridhandler L, et al: Ultracentrifugal characteristics of macrohype ramylase mia serum. Clin Chim Acta 35:299-304, 1971. 3. Levitt MD, Goetz! EJ, Cooperband SR: Two forms of macroamylasemia. Lancet 1:957-958, 1968
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4. Wilding P, Geokas MC, Haverback BJ, et al: Macroamylasemia due to protein-bound amylase. Am J Med 47:492-496, 1969 5. Bosseckert H, Winnefeld K, Seidel K: Macroamylasemia with paraproteinemia and the malabsorption syndrome. Ger Med Mon 14:133-135, 1969 6. Hedger RW, Hardison WGM: Transient macroamylasemia during an exacerbation of acute intermittent porphyria . Gastroenterolo gy 60:903-908, 1971 7. Hansen HR, Van Kley H, Knight WA: Macroamylasemia due to binding by protein. Am J Med 52:712-720, 1972 8. Tompkins RK, Adams, JR: Macroamylasemia in the post -operative patient. Arch Surg 105:630-632, 1972 9. Levitt MD, Duane WC, Cooperband SR: Study of macroamylase complexes. J Lab Clin M ed 80:414-422, 1973 10. Long WB, Kowlessar OD : A rapid thin layer test for macroamylase with observations on the values of macroamylase in six patients. Gastroenterology 63:564-571, 1972 11. Henderson AR, King J, Imrie CW: Anomalous response of macroamylase to assay temperature. Clin Chern 19:123-124, 1973 12 . Mutzbauer H, Schulz GV: Die bestimmung der molekularen Konstanten von a-amylase aus Humanspeichel. Biochim Biophys Acta 102:526-532, 1965 13. Fridhandler L, Berk JE: Macroamylasemia: rapid detection method. Clin Chern 17:423-426, 1971 14. Barrows D. Berk JR, Fridhandler L: Macroamylasemia-survey of prevalence in a mixed population. N Engl J Med 287:1352, 1972 15. Levitt MD, Rapoport M, Cooperband SR: Renal clearance of amylase in renal insufficiency, pancreatitis, and macroamylasemia . Ann Intern Med 71:919- 925, 1969 16. Blainey JD, Northam BE: Amylase excretion by the human kidney. Clin Sci 32:377-383, 1967 17. Duane WC, Frerichs R, Levitt MD: Distribution , turnover and mechanism of renal excretion of amylase in the baboon . J Clin Invest 50 :156- 165, 1971 18. Fridhandler L, Berk JE, Ueda M: Isolation and measurement of pancreatic amylase in human serum and urine. Clin Chern 18:1493-1497, 1972 19 . Berk JE, Fridhandler L, Montgomery K: Simulation of macroamylasemia by salivary-type ("S type") hyperamylasemia. Gut 14:726-729, 1973 20. Soll AH: A new approach to molecular configuration applied to aqueous pore transport. J Gen Physiol 50:2565-2578, 1967 21. Oken DE, Flamenbaum W : Micropuncture studies of proximal tubular albumin concentrations in normal and nephrotic rats. J Clin Invest 50:1498-1505, 1971
C'opHi ~ ht
©
IB7~
()7: J49- Jf>4. 1974 ll\' Tht· \l'illi"n" 8; \l'ilk ins (',_
Vo l. G7. !\In. I Prini<'d in U.S.A.
CASE REPORTS AN UNUSUAL FORM OF MACROAMYLASEMIA TEHRIEt. BEHGGRE:\ A:\ D
M ICI-IAF.t. D.
L Ev rrr
Depa rtment of MPdicin P. University of Minnesota Hospitals. Minneapolis. M innesota
The apparent molecula r weight of previously st udied macroa mylases is considerably larger than norma l se rum amylase, and th e existence of macroamyl ase has therefore been easi ly docum ented by crude gel filtration t ec hniques. This report desc rib es a patient with a persistantly elevated serum amylase level resulting from an unusual macroamylase which was only marginally larger than normaL The slightly increased size of this enzyme markedly inf1uenced its ren al clearance, and the existence of this macroa mylase was first suspected because of the patient 's consistently low C .\m: C cr ratio. Doc umentat ion of the increased size of this enzyme required careful gel filtrat ion study. Isoelectric focusing showed this macroamylase to have a lower isoelectric point than did the two major components of normal serum amylase. Th e develop m ent of mild pancreatitis after pan creatic duct cannulation provided a uniqu e opportunity to observe the serum and urin e a mylase values that occur when pancreatitis is superimposed on an underlyin g macroa mylase mi a . The existence of an abnormally large form of se rum amylase ("macroamylase") has been demonst rat ed in approxim ately 45 patients. t-t' Although apparently not injurious to health , m ac roamylase is nevertheless clinically important in that it usually causes hyperamylase mia, and failure to recogni ze this entity may result in an erroneo us di agnosis of pancreat ic disease . Di agnos is of this condition has been relatively simple since the large size of the macroamylase (gre ater than 100,000 mol wt in all cases) precludes its renal clea rance, and , as a result , the hyperamylasemia has been consistantly associated with a normal or low urinary a mylase. The Rece ived October 5, 1973 . Acc epted January 10. 1974. Address requests for repr ints to: Dr. Michael D. Le vi tt, Box 346, Un iversity of Minnesota . Depart me nt of M ed icin e, Medical Sc hool, Mayo Memorial Bu ildin g, Minn ea polis, Minn esot a 55455 . Supported by Nat ional Institut es of Health Grant 5 R01 AM1 3309. 149
large size differential between m acroamylase and normal amylase (mol wt of 55,000' 2 ) also makes it possible to easily document the presence of the large enzyme by simple gel filtration screening techniques. 13 . t • This report describes serial studies of serum and urinary amylase in a patient with a heretofore undesc ribed form of macroamy lase which was only fractionally larger than normal. The correct diagnosis was initially missed because she frequently had a sli ghtly elevated urin ary amylase, and the screening gel filtration test for macroamylase was negative. However, a persistently low renal clearance ratio of amylase to creatinine (C Am: C cr) suggested the presenc e of an abnorma ll y large amylase 15 which was then documented by more refined gel filtration studies. This case demonstrates the value of the simple meas urement of C Am : C cr in assessing hypera mylasemia. Of additional interest is the fact that the patient developed
CASE REPORTS
150
mild pancreatitis after pancreatic duct cannulation, thus providing a unique opportunity to study the changes in serum and urinary amylase which occur when pancreatitis is superimposed on an underlyin g macroamylasemia.
Case Report The patient, a 69-year-old housewife, was referred to the University of Minnesota Hos pitals for evaluation of abdominal pain associated with persistent hypera mylasemia, ra ngin g from 164 to 1800 Somogyi units during the 18- mon t h period prior to admission. For the previous 5 years, she ha d ha d one to three attacks eac h yea r of dull epigastric pa in often associated with vo miting . These attacks lasted 4 to 8 hr, a nd then cleared without specifi c therapy. She denied diarrhea, fever, weight loss, ja undi ce, or a pprec iable ethanol in gest ion. Because of easy fatiguability. she had spent much of the past 2 yea rs in bed. Physical exa mina t ion was entirely within normal limits . There was no evidence of muscl e wea kn ess , an d the easy fatiguability was thought to be of functional origin. Laboratory studies which were within normal limits includ ed: hemoglobin , white blood count , erythrocy te sed imen tat ion ra t e, urinal ys is, serum creat inine (0.9 per 100 ml) and creatinine clearance (104 ml per 1.73 m 2 ), se rum electrolytes. calcium. phosphorous. carotene, cholesterol. trigl~·cericle s, bilirubin. SGOT, a lka line phosphatase. a nd electrophores is . A glucose tol era nce test was no rmal, as was a n exercise electroca rdi ogram . A 4-dav stool collection showed 1.9 g. of fat per clay: Serum lipase was normal on two occasions. A rapid sc reenin g test for mac roa mylase usin g Sephadex G-100, as described by Friel ha ndler and Berk , 13 was negati\·e on two occas ions in that no a my lase activ ity was obsen·ed in th e fract ions conta inin g blue dextran. Chest X -ray a nd a fl at plate of th e abdo men were normal. An upper gastroint est in a l seri es re\·ea led a deformed duodenal bulb without e\·idence of a n act i\·e ulcer. The small bowel follO\\·-throu gh was normal. as was a barium enema. Endoscopy re\·ea led a normal sto mac h a nd duo de num . a nd cannulation of th e s phin cte r of Odcli with retrograde injection of contrast mat eri a l re\·ea led com pl etely norm a l pancrea ti c an d bilian· ducts and a normal ga llbladder. During th~ inj ec tion oft he contrast material. the patie nt complained of se\·ere epigast ric pa in. which. along with assoc iat ed hypotension. cleared in abo ut 5 min. Aft er cannula-
Vol . 67, No.1
tion s he had a mild epigastric pa in , an elevated temperature ranging from 99 to 101 F and her previously norma l whit e blood count increased to 11 ,250 with 84o/r neutrophils. These symptoms were interpreted as being indicati\·e of mild pancreatitis , and th ey clea red after several d ays of n asogastric suction.
Studies of Serum and Urinary Amylase M ethods . Serum and urinary a my lase were assayed by a sacc harogenic technique using 3, 5-dinitrosalizylic acid as a reagent. On e unit of a mylase ac ti vity is defined as that a ctivit y which liberates 1 mg of maltose per hr at 37 C. One of these units per 1 ml is roughly comparable to 13 Somogyi units, and the upper limits of norm a l (mean + 2 SD) for serum a nd urinary a mylase (2-hr urine collection) a re 11.4 U per ml and 48 U per min , res pecti vely. 1 5 The ratio of CAm: Cc, was calculated using standard clearance formulae . 1 6 The serum sample for these calculations was obtained approximately at the midpoint of the 2-hr urine collection. The lower and upper limits of norma l are 1.1 and 4.1 %, respectively. 1 5 The apparent molecular size of amylase was determined by gel filtration of 1-ml samples on 2.5- by 40-cm columns packed with polyacrylamide (B io-Gel P-150) or Sephadex G-100 . Except when noted columns were equilibrated with and eluted with 0.02 M phosphat e buffered saline (pH 7 .0). To det ermine if the macroamylase dissociated at an acid pH , the sa mpl e was dialyzed against 0.02 M glycine-HCl buffer (pH 3.4) , and then was chromatographed on a polyacrylamide column equilibrated with this acid buffer. Blue dextran and cytochrome c were added to the sa mpl e to be chromatographed to serve , res pectively, as intern a l m ar kers of the void volum e and elution volume of a substance with molecular weight of 13,000, respectively. In order to compare and quantitate objectively the elution patt ern of a mylase from one column run to the next , the percentage of the total amylase activity eluting prior to the peak of s imultan eo us ly chromatographed cytochrome c (measured at optical density 400) was det ermin ed . Isoelectric fo cus ing was ca rried out on a 110-ml column using pH :3.5-10 carrier amph olyte (LKB Instruments. Stockholm , Sweden). Th e density gra di ent consisted of 0 to 50'i sucrose with a final a mphol yte concentration of Fi. The se rum to be focused was dialvzed against distilled water, a nd 1 ml of the s~rum was then applied to the column. Focusin g was carried out at 4 C for 48 hr . Approximately 60
CASE REPORTS
July 1974
fractions were coll ected , and each fraction was analyzed for amylase, pH, and protein (optical density 280).
Results Serial measurements of serum amylase , urinary amylase, and C Am: C cr are graphically shown in figure 1. The serum amylase was persistently elevated, but it fluctuated from the upper limits of norm al to 9 times normal. The only episode of abdominal pain during this hospitalization occurred on October 11 and 12 after duct cannulation . Thus, there was no relation between abdominal symptoms and the serum amylase level. Urinary amylase ranged from high normal to slightly greater than normal , with the exception of the two greatly elevated values obtained after the duct cannulation on September 11, 1972. With the exception of the two postcannulation values, the CAm: C cr was always below normal levels. The apparent development of pancreatitis after cannulation resulted in a dramatic rise of C.:.. m: C cr to supranormal levels. Isoelectric focusing (fig . 2) of five serum samples from normal subjects showed that serum amylase normally has two major components with isoelectric points of ap-
151
proximately 5.3 and 5.9, respectively . Three of the serum samples had a small shoulder at a pH of about 4.8 on the pH 5.3 peak (see fi g. 2). In contrast , the bulk of the patient's serum amylase had an isoel ectrical point of about 4.7 with minor peaks at 5.3 and 5.8 (fig. 2B) . Gel filtration patterns on polyacryla mide of amylase activity of normal serum , a typical macroamylase serum, and the patient's serum are shown in figure 3. The peak concentration of albumin , blue dextran , and cytochrome c are indicated by arrows . Normal serum amylase (fig. 3A) elutes slightly faster than cytochrome c in a relatively symmetrical peak. Typical macroamylase serum (fig. 3B) has an amylase component which elutes in the void volume . As shown in fi gure 3C, the patient's serum amylase peak was asymmetrical , and a larger proportion of the patient's serum amylase appeared to elute prior to cytochrome c than was the case with normal serum amylase, suggesting that a portion of her amylase was larger than normal. In order to determine objectively whether this was a reproducible finding , five different samples of the patient's serum and four different normal sera were chromatographed and t he frac-
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dotted lines .
152
CASE REPORTS
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FRACTION FIG . 2 . Isoel ect ri c foc usin g of th e amylase of a ty pical norm al serum (A) a nd th e patient ·s serum (B ) . Th e isoelectric points of t he a mylase peaks are indi cat ed. Norm al serum has two major co mponent s wi th isoelectric points of approxim ately 5.3 and 5.9. T he bulk of t he pati ent 's serum amy las e has an isoelect ric point of roughly 4.7 , with two small peaks a t the isoelectric points of normal serum amylase .
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Fract ion FIG . 3. Gel filtr a tion elut ion pa ttern on polyacryla mide of amylase from: A , normal se rum; B, ty pical mac roamylase serum ; a nd C. the se rum of the patient described in this pa per. Th e peak concentrations of blue dextran , albumin , a nd cy tochrome c are shown by arrows.
tion of the total amylase activity eluting ahead of the cytochrome c peak was calculated. A mean of 81 ± 3.4% (1 SEM) of the patient's serum amylase eluted before the
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cytochrome cas compared with 62 ± 2.1% of normal serum amylase, a significant (P < 0.01) difference. The difference in elution patterns between normal serum amylase and the patient's serum amylase was also observed when gel filtration was carried out on Sephadex G-100. Gel filtration may markedly underestimate the quantity and perhaps the apparent size of the macroamylase complex since some complexes dissociate during gel filtration at neutral pH. 9 · 1 0 Long and Kowlessar 10 showed that increasing the volume of the sample to be chromatographed will tend to reduce this dissociation. The elution pattern of 5 ml of the patient's serum was virtually identical to that of 1 ml , suggesting that the pattern obtained with the 1-ml sample did not represent a much larger macroamylase which was rapidly dissociating during chromatography. When gel filtration was carried out at pH 3.4, the patient's serum amylase and normal amylase eluted in identical fashion , suggesting that the patient's macroamylase represented an acid dissociable complex. In an attempt to ascertain whether amylase was bound by a dialyzable material, a sample of serum was exhaustively dialyzed against pH 3.4 buffer . The pH of the sample was then raised to pH 7.0 with NaOH, incubated at 37 C for 1 hr, and gel filtered at pH 7 .0. The elution pattern of amylase was identical to that of the patient 's untreated serum (82% of amylase eluted prior to cytochrome c peak) indicating that the complex remained intact and that the binding factor had not passed into the dialysate . Several studies have suggested that salivary amylase is cleared more slowly by the kidney than is pancreatic amylase . 17 • ' 8 To determine whether the patient's serum amylase might represent her salivary amylase, saliva was diluted 1 :50 and chromatographed. The elution pattern was identical to that of normal serum amylase with 61 % eluting prior to the cytochrome c peak. Discussion This case further broadens the spectrum of the already heterogenous condition
July 1974
CASE REPORTS
known as macroamylasemia. The abnormal enzyme has been found in patients with a variety of disease states as well as normal subjects identified by routine screening. 14 As was the situation in this case, abdominal pain has been a common accompaniment ofmacroamylasemia. This relation may well be fortuitous, however, in that abdominal pain leads to measurement of serum amylase activity, thus uncovering an unrelated macroamylasemia. Although the cause of the abdominal pain in our patient was not elucidated, the normal pancreatic duct study makes it quite unlikely that she suffered from chronic pancreatitis. Berk et al. 19 have recently described 3 patients with an elevated serum amylase level, low renal CAm: C cr. and no obvious macroamylase in the serum. Ion exchange chromatography showed that the bulk of the serum amylase of these patients behaved like one of the two major amylase components of normal serum which they have termed "salivary type" amylase. In contrast, it is clear from isoelectric focusing studies that the amylase of our patient was not one of the major components of normal serum amylase. In addition, the possibility that her serum amylase was primarily salivary amylase was also excluded by gel filtration studies which showed that her serum amylase eluted more rapidly than did salivary amylase. The apparent molecular weights of previously studied macroamylases have ranged from 160,000 to greater than two million. 2 ' 3 Although the dissociation of macroamylase complexes makes it difficult to be certain ot molecular size, the macroamylase of our patient appeared to be marginally greater than that of normal amylase. Demonstration of this small size differential required careful comparison of the elution patterns of amylase and cytochrome c, and it is possible that similar cases have gone undetected. The minimally increased size of the patient's amylase did, however, reduce its rate of -clearance by the kidney, resulting in a consistently low renal CAm :Cc, (except for the values obtained after the pancreatic duct study). The renal excretion of amylase appears
153
to result from glomerular filtration without appreciable tubular reabsorption. 17 The dimensions of the glomerular "pore" are such that for molecules in the size range of amylase, minor differences in molecular radii result in large differences in glomerular permeability. For example, egg albumin, amylase , and serum albumin which have respective molecular weights of 45,000, 55,000, and 68,000, are filtered at rates of 22%, 20 2.5%, 15 and less than 0.1 %21 of the glomerular filtration rate. The permeability of the glomerulus to amylase is most readily expressed as the ratio of CAm: Cc,, a value which normalizes for individual differences in glomerular filtration rate. 15 ' 16 The finding of a low CAm: Cc, in a patient with ' an elevated serum amylase level usually suggests the presence of macroamylasemia since this ratio is consistently elevated to supranormal levels in acute pancreatitis. 15 One explanation for this increased clearance in pancreatitis is that pancreatic amylase is more rapidly cleared by the kidney than is salivary 17 • 18 or endogenous serum amylase. 17 As shown in figure 1, a dramatic increase in CAm: Ccr occurred when mild pancreatitis (after pancreatic duct cannulation) was superimposed on the preexisting macroamylasemia. Presumably in this situation, the amylase binding substance was completely saturated, and the free pancreatic amylase was rapidly cleared by the kidney. However, the possibility that pancreatitis decreased the binding of amylase could not be ruled out. The hallmark of macroamylasemia is a normal or low urinary amylase in the face of an elevated serum amylase. It is apparent from figure 1, however, that very high serum amylase levels in the absence of pancreatitis were associated with mildly elevated rates of urinary amylase excretion . This finding probably reflects the relatively small size of our patient's macroamylase, which made possible a slow but appreciable rate of glomerular filtration whereas the glomerulus is totally impermeable to the larger forms of macroamylase. The high serum amylase levels in macroamylasemia are thought to be the result of slow metabolic clearance of the abnor-
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CASE REPORTS
mal enzyme, rather than excessive deposition of amylase into the blood, as is the case in pancreatitis. The question thus arises as to how our patient could excrete excess quantities of amylase in her urine , if, presumably, amylase was entering her serum at a normal rate. One explanation is that studies in baboons have shown that, of the total metabolic clearance of serum amylase, only about 20% is removed in the urine while 80% is cleared by extrarenal mechanisms. 17 If the slightly increased size of the patient 's amylase hindered extrarenal clearance to a greater extent than renal clearance, the urinary amylase could be elevated even though normal quantities of amylase were entering the serum. The elution pattern of the patient's serum amylase was indistinguishable from that of normal serum amylase after treatment with an acid pH, suggesting that her macroamylase represented an acid dissociable complex of normal-sized amylase bound to an abnormal substance. These amylase-binding substances have been shown to be IgA in 2 cases, 2 IgG in 1 case, 7 and apparently something other than immunoglobulin in the bulk of the cases. The slight difference in size between the macroamylase in this case and normal amylase indicate that the binding substance was smaller than the immunoglobulins. Presumably, the serum amylase level in macroamylasemia is largely determined by the amount of binding substance in the serum. The wide swings of serum amylase levels observed in our patient over a period of several days differs from most previous cases of macroamylasemia, and suggests that there were rapid fluctuations in the serum concentration of this amylase-binding substance. REFERENCES 1. Wilding P, Cooke WT, Nicholson GI: Globulin-
bound amylase: cause of persistently elevated levels in serum. Ann Intern Med 60:1053-1059, 1964 2. Ueda M, Berk JE, Fridhandler L, et al: Ultracentrifugal characteristics of macrohype ramylase mia serum. Clin Chim Acta 35:299-304, 1971. 3. Levitt MD, Goetz! EJ, Cooperband SR: Two forms of macroamylasemia. Lancet 1:957-958, 1968
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4. Wilding P, Geokas MC, Haverback BJ, et al: Macroamylasemia due to protein-bound amylase. Am J Med 47:492-496, 1969 5. Bosseckert H, Winnefeld K, Seidel K: Macroamylasemia with paraproteinemia and the malabsorption syndrome. Ger Med Mon 14:133-135, 1969 6. Hedger RW, Hardison WGM: Transient macroamylasemia during an exacerbation of acute intermittent porphyria . Gastroenterolo gy 60:903-908, 1971 7. Hansen HR, Van Kley H, Knight WA: Macroamylasemia due to binding by protein. Am J Med 52:712-720, 1972 8. Tompkins RK, Adams, JR: Macroamylasemia in the post -operative patient. Arch Surg 105:630-632, 1972 9. Levitt MD, Duane WC, Cooperband SR: Study of macroamylase complexes. J Lab Clin M ed 80:414-422, 1973 10. Long WB, Kowlessar OD : A rapid thin layer test for macroamylase with observations on the values of macroamylase in six patients. Gastroenterology 63:564-571, 1972 11. Henderson AR, King J, Imrie CW: Anomalous response of macroamylase to assay temperature. Clin Chern 19:123-124, 1973 12 . Mutzbauer H, Schulz GV: Die bestimmung der molekularen Konstanten von a-amylase aus Humanspeichel. Biochim Biophys Acta 102:526-532, 1965 13. Fridhandler L, Berk JE: Macroamylasemia: rapid detection method. Clin Chern 17:423-426, 1971 14. Barrows D. Berk JR, Fridhandler L: Macroamylasemia-survey of prevalence in a mixed population. N Engl J Med 287:1352, 1972 15. Levitt MD, Rapoport M, Cooperband SR: Renal clearance of amylase in renal insufficiency, pancreatitis, and macroamylasemia . Ann Intern Med 71:919- 925, 1969 16. Blainey JD, Northam BE: Amylase excretion by the human kidney. Clin Sci 32:377-383, 1967 17. Duane WC, Frerichs R, Levitt MD: Distribution , turnover and mechanism of renal excretion of amylase in the baboon . J Clin Invest 50 :156- 165, 1971 18. Fridhandler L, Berk JE, Ueda M: Isolation and measurement of pancreatic amylase in human serum and urine. Clin Chern 18:1493-1497, 1972 19 . Berk JE, Fridhandler L, Montgomery K: Simulation of macroamylasemia by salivary-type ("S type") hyperamylasemia. Gut 14:726-729, 1973 20. Soll AH: A new approach to molecular configuration applied to aqueous pore transport. J Gen Physiol 50:2565-2578, 1967 21. Oken DE, Flamenbaum W : Micropuncture studies of proximal tubular albumin concentrations in normal and nephrotic rats. J Clin Invest 50:1498-1505, 1971