Table 6. N o r m a l values for the enzyme activity in cerebrospinal fluid
Enzyme** Method Unit Author N u m b e r
of cases N o r m a l values S D G O T 28) Wroblewski units/ml. Liebermann
2
> 44 12.1 ( 5 - 2 4 ) ± 5 . 4 9 28) Wroblewski units/ml. Katzman 8) 45 11.9 ( 5 - 2 1 )
29) Wroblewski units/ml. Manai**) 29 11.1 ± 4 . 9 6
G P T 29) Wroblewski units/ml. Manai^) 29 3 ± 2 . 7 4
L D H 30) [xmoles/hr./ml.; 37° C Brutish 14 1.07 ( 0 . 6 7 - 1 . 6 7 ) PHI 31) umoles/hr./ml.;
pH 7.8; 37°C
Bruns
34
) 28 0 . 9 ( 0 . 1 5 - 2 . 3 )
R5P-I 32) u.moles/hr./ml.;
p H 7 . 5 ; 3 7 ° C
Bruns
34
) 16 0.07 ( 0 . 0 3 - 0 . 0 8 )
I C D H 33) m(jLmoles/hr./ml.;
25° C
van Rymenant
23
) 42 6 . 2 ( 1 . 7 - 1 1 . 9 ) ± 2 . 5
Aldolase and glyceraldehyde-3-phosphate dehydrogenase were not detectable after incubation for 1 hr. at 37°C34).
*) G O T = Glutamate-oxalacetate transaminase; G P T = Phosphohexoseisomerase; R5P-I = Ri-bose-5-phosphate isomerase; I C D H = Isocitric dehydrogenase.
III. Enzyme Activity in Urine
Just as enzymes can appear in blood and in other body fluids so can they also appear in bile and urine. As early as 1846 Magendle
1
) detected amylase in urine. Since then numerous enzymes have been found in urine, although under normal conditions only those with molecular weights of less than about 60000 are found
2
*.
Even in animal experiments and in patients with high enzyme activity in serum there was n o3 - 5 ) or only a very slight
6 9
* excretion of LDH, GOT, GPT and ALD in urine, providing that the kidney function was intact. Amelung et al.® suggest that the cell debris of the ex
cretory urinary passages is the source of the slight enzyme activity found in normal urine.
28) A. Karmen, J. clin. Invest. 34, 131 [1955].
29) /v. E. Tonhazy, U. G. White and W. W. Umbreit, Arch. Biochem. Biophysics 28, 36 T1950].
30) F. Kubowitz and P. Ott, Biochem. Z. 314, 94 [1943].
3D F. H. Brims and K. Hinsberg, Biochem. Z. 325, 532 [1954].
32) F. H. Bruns, Biochem. Z. 327, 523 [1956].
33) S. K. Wolfson jr. and H. G. Williams-Ashman, Proc. Soc. exp. Biol. Med. 96, 231 [1957].
34) F. H. Bruns, L. Jakob and F. Weverinck, Clin. chim. Acta /, 63 [1956].
1) M. Magendie, C. R. hebd. Seances Acad. Sci. 23, 189 [1846].
2) R. Richterich: Enzymopathologie. Springer, Heidelberg 1958, p. 589.
3) M. West and H. J. Zimmermann, J. Lab. clin. Med. 52, 185 [1958].
4) M. Dunn, J. Martins and K. R. Reisman, J. Lab. clin. Med. 51, 259 [1958].
5) D. Klaus, Klin. Wschr. 36, 207 [1958].
6) E. Kemp and T. Laursen, Scand. J. clin. Lab. Invest. 12, 463 [I960].
7) S. B. Rosalki and / . H. Wilkinson, Lancet 327 [1959].
8) D. Amelung, H. D. Horn and E. Schroder, Klin. Wschr. 36, 963 [1958].
9) M. Chinsky, G. L. Shmagranoff and S. Sherry, J. Lab. clin. Med. 47, 108 [1956].
700 Section C : Measurement of Enzyme Activity
A raised enzyme level in serum has been occasionally found in kidney damage
3
»
9 - 1 5 )
. In contrast, an increase of enzyme activity in urine was detectable in the majority of cases5
'8
'
1 6
*. Kidney infarcts in experimental animals led to increased levels of LDH in serum and urine
6
' 1 6 , 1 7 ) .This demonstrates that the enzyme activity in urine is independent of the enzyme level in blood
8 )
. Kemp and Laursen® and Rosalki and Wilkinson
1
) found no significant proportional
ity to the extent of proteinuria, while Klaus
5
) found only a loose one and Crockson
1
®
found a significant one. Crockson showed by histological studies of kidney punctures, that on the average, higher activity of LDH occurred in urine when there was severe kidney damage.
There are differing views on the question, whether the enzymes detected in urine in kidney damage originate from the kidney cells which are destroyed or whether they result from a disturbance of the filtration efficiency. To settle this point the clearance rates of various serum proteins and enzymes have been compared
5
>
1 8 )
. Crockson1
® found a significant relationship in 61 examinations between LDH and albumin clearance with a quotient of 70:100. The value of this quotient corresponds to the ratio of the molecular weights. Worthy of particular attention are the studies of Kemp and Laursen
6
) on the LDH isoenzymes in serum and urine after ischaemia of the kidneys with or without injection of crystalline en
zymes. After electrophoresis of the serum proteins they found LDH activity in the a and (3-globulin fraction. The LDH activity in urine usually only occurred in the a-globulin frac
tion. Only after recirculation through the kidneys and injection of renin, which damages the membrane of the glomeruli
1 9
), could two LDH fractions be demonstrated in urine with mobilities corresponding to a and (3-globulin. It is probable that further studies on the distri
bution of the enzymes detectable in urine after kidney damage may lead to a significant improvement of the diagnosis of kidney diseases.
IV. Notes on the Technique of Enzyme Assays
The critical evaluation and comparison of data published by various authors is made difficult less by the choice of different units, than by differences in the methods used and especially by the various definitions of the stages of the diseases for which the characteristic changes have been described. Generally such data can only be compared directly if they were obtained with the same experimental conditions. Fig. 12 shows with reference to LDH, how different substrate concentrations lead to considerable differences in the measured values. Differences in pH, temperature, etc. may partly reduce the differences or possibly they may increase them.
It is difficult to lay down optimum substrate concentrations for enzyme assays, as enzymes of the same specificity from different organs (isoenzymes) exhibit different substrate affinities and also as the proportion of the isoenzymes in the plasma is continually changing. According to the type of diseased organ and according to the course of the disease different substrate
io) F. Wroblewski, Advances clin. Chem. 7, 93 [1958].
ID L. P. White, N e w England J. Med. 255, 984 [1956].
12) W. E. C. Wacker, D. D. Ulmer and B. L. Vallee, N e w England J. Med. 285, 449 [1956].
13) K. M. Hsieh and H. T. Blumenthal, Proc. Soc. exp. Biol. Med. 91, 626 [1956].
14) H. J. Zimmermann and H. G. Weinstein, J. Lab. clin. Med. 48, 607 [1956].
is) J. S. LaDue and F. Wroblewski, Circulation 11, 871 [1955].
16) M. M. Bett, J. D. Skaggs, G. Johnston and F. B. Herseley, Surg. Forum 9, 65 [1959].
17
) E. Kemp, Acta path, microbiol. scand. 45, 1 [1959].
is) R. A. Crockson, Lancet 140 [1961].
19) A. L. Sellers, S. Smith, J. Marmorston and C. Goodman, J. exp. Med. 96, 643 [1952].
L a In Medicine 701
concentrations must be used, in order to judge exactly the increase in activity and the durat
ion of the increase. As in practice this is impossible, well-tried methods should only be changed for the purpose of clinical comparison when a real advance will result.
WroblewskiGta\.U-$ j Norn et a l .
2)
I Schmidt et al.
3
*
Fig. 12. Dependence of the L D H activity in serum o n the pyruvate concentration.
— — Carcinoma Infarction
— • — • — Cirrhosis
— . . — Pernicious anaemia
— . . . — Hepatitis
T o make the Figure clearer only half the measu
red L D H activity has been plotted for pernicious anaemia and hepatitis.
4 5 10-3 M Pyruvate t-Engelhard-Golkel et al.
6
* Lo'hr et al.
5
)
— Hauss et al.
4
*
The arrows indicate the approximate pyruvate concentration used by the authors.
With the multiplicity of enzymes in serum it is necessary to confirm that the assay method for a particular enzyme only measures the activity of that enzyme and that the assay is not interfered with by a second enzyme. For example, it is frequently not appreciated that on addition of keto acid in the assay of transaminases a GIDH catalysed reaction is simultane
ously started. Although usually very little GIDH is found in serum and therefore this interference can often be ignored, sometimes, for example, in obstructive jaundice relatively high GIDH activity is found temporarily in serum
7 )
. Consideration of interference from this source (refer to p. 841, 851) is therefore necessary.
The substrates of the reactions catalysed by enzymes are frequently found in variable and significant amounts in the body fluids. This fact is usually taken into account in the enzyme assays, since the serum is pre-incubated with the coenzyme and the assay is only started after the reaction of the endogenous substrates. However, due to the variable concen
trations of the endogenous substrates different amounts of coenzyme are consumed, so that in extreme cases the concentration of coenzyme is no longer optimal. On the other hand, the reaction can be inhibited by too high a concentration of coenzyme {e.g. GIDH). It is not
1) F. Wroblewski and / . S. LaDue, Proc. Soc. exp. Biol. Med. 90, 210 [1955].
2) H. D. Horn and D. Amelung, Dtsch. med. Wschr. 82, 619 [1957].
3) E. Schmidt, F. W. Schmidt and E. Wildhirt, Klin. Wschr. 36, 172, 227 [1958].
4
> W. Hauss and H. J. Leppelmann, Klin. Wschr. 35, 65 [1957].
G. W. Lohr, H. D. Waller and O. Karges, Klin. Sschr. 35, 871 [1957].
6) A. Engelhardt-Golkel, R. Lobel, W. Seitz and I. Waller, Klin. Wschr. 36, 462 [1958].
?
) E. Schmidt and F. W. Schmidt: Verhandlungen I. Europaisches Symposium med. Enzymologie, Milan 1960. S. Karger, Basle 1960, p. 100.
702 Section C: Measurement of Enzyme Activity
certain that these, often decisive, alterations of the reaction conditions are always given due consideration in routine assays of enzyme activity. A deficiency of coenzyme cannot be detected directly in the assay of enzyme activity by means of colour reactions.
Another variable with activating or inhibitory effects on the enzyme activity is the content of ions and other substances in the material to be examined
8
*. This makes the comparison of enzyme activity in diseases of different organs and in different stages of disease difficult;
after dilution of the serum the enzyme activity may not decrease in direct proportion to the dilution. The addition of chelating agents to bind certain ions can cause an increase in activity of certain enzymes, but it may inhibit other enzymes. The strong inhibitory action of heparin