Method for measuring plasma diamine oxidase

CORRESPONDENCE

Method for measuring diamine oxidase

plasma

for choriocarcinoma and hydatidiform mole from nor-ma1 pregnancy. In these categories the plasma diamine oxidase assay was found to be a sensitive and reliable indicator of the status of pregnancy. “High-risk” pregnancies, as described by Southren and associatesll-*s are those in which the development of plasma DA0 activity is relatively low (100-300 units) as compared to normal pregnancies (500-5,000 units). A careful evaluation of the method of Resnik and Levine in our laboratory showed that the primary reason for their failure to confirm this observation is attributable to their modifications of our procedure which reduced the sensitivity of the original method at least tenfold. This reduction in sensitivity is due to their use of both a smaller volume of plasma, resulting in a decreased yield of actual radioactive product, and an increased buffer concentration which is inhibitory to the enzyme activity. Table I shows the low sample c.p.m./blank c.p.m. ratio in the method of Resnik and Levine as compared to that of Southren and associates. This indicates the markedly reduced sensitivity of Resnik and Levine’s method. Fig. 1 shows the competitive inhibition of the increased buffer concentration on DA0 activity. The resultant decreased sensitivity is most apparent at low Ievels of plasma DA0 activity. It therefore, would be most difficult, using the method of Resnik and Levine, to detect low plasma DA0 pregnancies or to define the lower limits of normal pregnancy. Thus, as indicated by Resnik and

To the Editor: This letter refers to the article by Resnik and Levine entitled “Plasma diamine oxidase in pregnancy; a reappraisal,” which was published in the August 1, 1969, issue of the AMERICAN JOURNAL

OF OBSTETRICS

AND

GYNECOLOGY.

We

feel it necessary, because of some obvious and other more subtle errors in biochemical technique and scientific method, to comment in detail on their article. Historically, the plasma diamine oxidase (DAO) assay was proposed as an indicator of human pregnancy, because of its marked rise during gestation, by Marcou and co-workers.1 in 1938. This observation was confirmed by other investigators.21 s Moreover, additional studies suggested a relationship between low levels of plasma diamine oxidase and abnormal pregnancy.*-7 However, the assay failed in its clinical application because of the inherent lack of sensitivity of the methods available at the time. Renewed interest in plasma diamine oxidase for the study of pregnancy was stimulated by the sensitive and highly specific radioassay of Okuyama and Kobayashi.8 Southren, Kobayashi, and co-workers,Q-*s using a modification of their original procedure, demonstrated the usefulness of the radioassay for plasma diamine oxidase as (a) a pregnancy test, (b) a monitor of fetal development, (c) a monitor of “high-risk” pregnancies, and (d) as a possible differential diagnostic test

Table I. A comparison of the sensitivity of the methods of Resnik and Levine (R and L) and Southren et al. (S et al.) for detecting plasma diamine oxidase (DAO) in nonpregnant subjects and in “high-risk” pregnancy

DA0 Plasma

Nonpregnant High-risk pregnancy

units

(range)

o- 10 100-300

Net c.p.m. R and L ) S et al. o- 13 o- 102 127-383

1,021-3,061

Blank (c.p.m.) R and L 1 S et al. 54 53 54

1246

53

sample c.p.m. blank c.p.m. R and L 1 S et al. o-o.2 o- 2.

Range of

2.4-7.1

19.3-57.8

Volume Number

106

Correspondence

1247

8

Levine, it is frequently not possible to differentiate pregnancy from the nonpregnant state. Had Resnik and Levine used the method of Southren and associates as described, without modifications, their results would likely have been comparable to those already in the literature. The authors indicate on page 1062 of their paper that if a direct conversion of their units to the DA0 units of Southren and associates was possible, 17.6 mpmoles per milliliter of Resnik and Levine would be equivalent to 1 DA0 unit of Southren and associates. Our calculation, however, indicates that if this were so the values reported for their normal curve shown in Fig. 1 would be in the nonpregnant range as reported by Southren et al. If the authors would recalculate their ratio, they would find that their stated theoretical equivalency should be reversed; i.e., 17.6 DA0 units equals 1 mpmole of putrescine metabolized per milliliter plasma. The normal curve of plasma DA0 values shown by Resnik and Levine14 in their Fig. 1 presents a single plasma DA0 value from each of 110 patients with “normal” pregnancy. The authors do not state whether the patients included in the figure have normal obstetrical histories. We have found that patients with poor obstetrical histories may have abnormally low curves despite the successful termination of pregnancy.ll-1s We have indicated in several publications that we consider these abnormal curves to define a group of biochemical “high-risk” pregnancies. It is out of this group that we find

THEDWBLE-RECIPRCCALORLINEWEAVER-BURK PLOTOF+VERSUS& DEMONSTRATING COMPETITIVE INHIBITION OF DIAMINE OXIDASE ACTIVITY BY THE INCREASED BUFFERCONCENTRATION USED INTHE METHOD OF RESNIK AND LEVINE.

!@I ‘/v

-10 -6 -6

-2

METHODOF RESNIK AND LEVINE.

0 2 4 6 6 IO 12 14 I6 I6 20

the problem pregnancies and the fetal wastage. We have also stressed the need for serial studies in our papers, a fact which was almost completely neglected in the publication of Resnik and Levine. In Figs. 2 and 4 of their paper, Resnik and Levine’4 present one plasma DA0 value from each of 11 patients with clinical “high-risk” pregnancies. There was a total of six values from 6 patients with normal pregnancies resulting in premature infants, and one value each from 5 patients with 5 different clinical categories of “high-risk” pregnancies. From this very sparse data the authors arrive at the conclusion that there is no relationship between the “high-risk” status and plasma DA0 values. Their data does not justify this conclusion. The method used is too insensitive to accurately define the low DA0 group. In any event, single determinations in such diverse entities as alcoholism, anemia, erythroblastosis, diabetes mellitus, etc., cannot provide sufficient data for the conclusions recorded. Moreover, it should be noted that we have not to date reported our findings on most of the clinical entities which the authors report. Therefore, there also can be no comparison of the clinical data. In conclusion, it is not possible to compare both methods since they are markedly different in sensitivity. The modifications offered by Drs. Resnik and Levine do not offer any improvement in the technique for measuring plasma DA0 and in fact may result in misleading data. One of our objectives from the very beginning in introducing the radioassay for plasma DA0 was to improve the sensitivity over the methods previously reported. In addition, the data presented by Drs. Resnik and Levine are sparse and do not justify their conclusion that the assay of plasma DA0 is not a reliable indicator of “high-risk” pregnancy. A. Louis Southren, Allan B. Weingold, New York Medical College Metropolitan Hospital Center New York, New York Yutaka Kobayashi, Worcester Foundation for Experimental Biology Shrewsbury, Massachusetts

M.D. M.D.

Ph.D.

REFERENCES V= VELQCITY OFENZYMATIC REACTION [S]=CONCENTRATION OFPUTRESCINE AS SUBSTRATE

Fig. 1.

1. Marcou, I., Athanasiu-Vergu, E., Chiriceanu, D., Cosma, G., Gingold, N., and Parhon, C. G.: Presse Med. 46: 371, 1938.

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2.

April 15, 1970 Amer. J. Obstet. Gynee.

Correspondence

Werle,

E., and

Effkemann,

G.:

Arch.

Gynaek.

170: 82, 1940. 3. 4. 5. 6.

7.

8. 9.

10.

11.

12.

Zeller, E. A., and Birkhauser, H.: Schweiz. Med. Wschr. 70: 975, 1940. Labhardt, A.: Mschr. Geburtsh. Gynaek. 112: 1, 1941. Ahlmark, A.: Acta Physiol. Stand. 9: 107, 1944. (Suppl. 28.) Anrep, G. V., Barsoum, G. A,, and Ibrahim, A.: J. Obst. Gynaec. Brit. Emp. 54: 619, 1947. Ahlmark, A., and Werko, L.: Toxemia of Pregnancy, A Ciba Foundation Symposium, Philadelphia, 1950, The Blakiston Company, p. 247. Okuyama, T., and Kobayashi, Y.: Arch. Biothem. 95: 242, 1961. Southren, A. L., Kobayashi, Y., Sherman, D. H., Levine, L., Gordon, G., and Weingold, A. B.: AMER. .T. OBSTET. GYNEC. 89: 199, 1964. Southren, A. L., Kobayashi, Y., Carmody, N. Cl.. and Weinaold. A. B.: AMER. J. OBSTET. G;NEC. 95: 615, i966. Southren, A. L., Kobayashi, Y., Weingold, A. B., and Carmody, N. C.: AMER. J. OBSTET. GYNEC. 96: 502, 1966. Southren, A. L., Weingold, A. B., Kobayashi, Y., Sherman, D. H., Grimaldi, R., and Gold, E. M.: AMER. T. OBSTET. GYNEC. 101: 899. 1968. Southren, A. L., and Weingold, A. B.: AMER. J. OBSTET. GYNEC. In press, 1970. Resnik, R., and Levine, R. J.: AMER. J. OBSTET. GYNEC. 104: 1061, 1969. ”

13. 14.

Reply to Drs. and Kobayashi To

Southren,

Weingold,

the Editor: In response to the letter by Drs. Southren, Weingold, and Kobayashi in which they comment on our article “Plasma Diamine Oxidase in Pregnancy : a Reappraisal” (AMER. J. OBSTET. GYNEC. 104: 1061, 1969): First, we acknowledge gratefully their pointing out our error in stating that 17.6 mpmoles of putrescine deaminated per hour equals 1 DA0 unit; actually, as they observed, the reverse is true. Next, we were surprised to learn that DA0 could be inhibited by phosphate buffer. We have checked this observation in our laboratory and found that at very low concentrations of substrate there is indeed a small degree of inhibition by phosphate which nearly disappears with increasing substrate concentration. Undoubtedly, this accounts for the observation stated in our paper that under our conditions of incubation 50 cg of substrate was inadequate to support maximal

enzyme reaction rates. The inhibition by 0.5M phosphate buffer when 105 pg of substrate are used (as in our assay) is negligible. As they indicated our method is less sensitive than is theirs largely because we used less plasma. If increased sensitivity is desired, the amount of plasma added can easily be increased. We made no effort to increase sensitivity in the course of studies reported in our paper because most DA0 values were high-easily within the range of reliable assay. We did not know until the studies had been completed which of the patients would fall in the abnormal or “high-risk” group. While low sensitivity might account for some of the low values in normal pregnancies it certainly would not account for the finding of high values in most patients with “high-risk” pregnancies. Increasing the sensitivity of the assay would only have accomplished a further apparent increase in DA0 activities in that group. Therefore, WC continue to feel that our data support our rather conservatively stated conclusion: “. . . assay of DA0 activity in plasma, as done in this laboratory, is not a reliable procedure for detection of the ‘high-risk’ pregnancy.” We acknowledge that we did very few serial studies of DA0 activities in the course of pregnancy. However, even our limited data indicate that there may be substantial upward or downward variations in the course of apparently normal uncomplicated pregnancies. This finding further undermines our confidence in the value of serial DA0 assays for detection of the “highrisk” pregnancy. Robert Robert Yale University School New Haven, Connecticut

Neutrophil pregnancy To

Resnik, 1. Levine,

M.D. M.D.

of Medicine

06510

hypersegmentcation

in

the Editors: In their paper “Neutrophil hypersegmentation and folic acid deficiency in pregnancy” (AMER. J. OBSTET. GYNEC. 104: 1163, August 15, 1969), Dr. Kitay and his co-workers quote Dr. V. Herbert as indicating that, besides acid folic and/or vitamin B,, deficiency, the only other conditions associated with neutrophil hypersegmentation are chronic renal disease and congenital hypenegmentation. Very long ago I found that thyro-