(Biological Research Laboratories, Harvard School of Dental Medicine; Department of Pharmacology, Harvard Medical School, Bo8ton,Massachusetts; and Graduate Department of Biochemi8try, Brandeis University, Waltham, Ma.@sachu@etts)

SUMMARY

The hypothesis that certain nonparathyroid tumors associated with hypercalcemiacontain a parathyroid hormone-like substance was examined experimentally. Biologic assays of extracts of the tumors in parathyroidectomized rats were negative orequivocal; factors involved in interpretation of the assays are discussed. Earlierpublished estimates of parathyroid hormone-like material in tumors, obtained by immunoassay of urea extracts of the tumors by a method utilizing direct complementfixation, were found to be too high because of a nonspecific reaction of the urea extracts.New evidence supporting the presence of parathyroid hormone-like material in hypercalcemic tumors was provided by data showing that HC1 extracts of the tumors, likeHC1 extracts of parathyroid glands, specifically inhibit the direct complement fixationby purified bovine parathyroid hormone and anti-bovine parathyroid hormone.Factors that restrict the inhibition reaction to qualitative identification of parathyroid hormone-like substances are discussed.

In recent years attention has been drawn to a syndromein patients with malignant neoplasms that is characterizedby marked hypercalcemia without gross or microscopicevidence of parathyroid enlargement or hyperactivity andwithout invasion of osseous tissue. In the majority ofcases, removal of the tumor was associated with the promptreturn of serum calcium to normal, whereas recurrence ofthe tumor was followed by reappearance of hypercalcemia(4). To explain these observations, it has been hypothesized that a blood-borne osteolytic substance is producedand released by the tumor and that this substance is similar or identical in chemical structure to parathyroid hormone. We first became interested in the syndrome andthe hypothesis in 1956, when Dr. John Eager Howardwrote one of us (P. L. M.) to inquire about the possibilityof testing extracts of hypercalcemic tumors for parathyroidhormone-like activity in our bioassay method in parathyroidectomized rats.

Parathyroid hormone-like activity in the tumor mightescape detection by biologic assay if the concentration ofthe activity were too low to meet the minimum dose requirement of the assay. Yet a low concentration of ac

1 Contribution No. 372 of the Graduate Department of Bio

chemistry, Brandeis University, Waltham, Mass.This investigation was supported in part by research grants

from the National Institute of Arthritis and Metabolic Diseases(A-1787), USPHS, and the American Cancer Society (E-222).

2 Special Research Fellow, National Institute of Arthritis and

Metabolic Diseases, USPHS.8 American Cancer Society Professor of Biochemistry, Brandeis

University, Waltham, Mass.

tivity would not be inconsistent with a high, clinicallysignificant rate of release of an active substance, owing tothe size of the tumor relative to that of normal or hyperactive parathyroid glands. The concentration of hormonein bovine parathyroid glands is a useful point of reference.The average fresh weight of 1 bovine gland is 120 mg (determined by weighing 900 carefully dissected glands).The concentration of parathyroid hormone in these glands,as estimated by bioassay of the classic hot dilute HC1 cxtract, is approximately 1600 units/gm or 200 units/gland.4The extract, which is customarily prepared with 10 ml ofacid/gm of glands, contains about 160 units/mi.

The biologic assay method (7), in which rats on a lowcalcium diet are parathyroidectomized and immediatelythereafter injected s.c. with a standard or unknown preparation, requires a minimum dose of 10—15units/rat toproduce a statistically significant elevation above the control level of serum calcium in a group of 5 test rats. Therefore, injection of 0.1 ml of bovine parathyroid extractnormally would yield a detectable effect. (Limited testsof extracts of normal human parathyroid glands and adenomas indicate that they do not contain any higher concentration and may contain a lower concentration of hormone than bovine glands.) In order for a similar extractof tumor to produce a detectable effect in our test rats at

4 Unpublished observations by P. L. Munson and B. A. San

born. The lower concentration reported by Kenny et al (6) wasforundissectedglands as receivedfrom the supplier. Whenproperallowance is made for nonparathyroid tissue in undissected glands,the 2 reports are in reasonable agreement.

1062

Evidence for Parathyroid Hormone in Nonparathyroid TumorsAssociated with Hypercalcemia1

PAUL L. MUNSON, ARMEN H. TASHJIAN, JR.,2@ LAWRENCE LEVINE@

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TREATMENTEXPERIMENT

IEXPERIMENTIIPlasma

calcium0(mg/lOO ml)Urine

P,@(mg/rat/hr)Plasma calcium'

(mg/lOOml)HC1

controlCysteinecontrolParathyroid extract, 15 USPunits

Parathyroidextract,60USPunits

Tumor No. 2 extractb6.1

7.17.1

11.1

7.80.14

0.110.43

1.22

0.156.0

7.18.3

12.1

8.3

MUNSON et al.—Parathyroid Hormone in Tumors 1063

the usual injection volume of 1.0 ml, the concentration ofhormone in the tumor would need to be one-tenth as highas it is in bovine glands, or if 4.0 ml, an excessive volume,were injected, the concentration of hormone would need tobe 0.025 as high as in parathyroid glands.

There are several other considerations to be taken intoaccount in the interpretation of results of a biologic assayfor parathyroid hormone (or any other active agent). Onethat is particularly relevant when testing a crude extractsuch as that of a tumor, in which the concentration ofparathyroid hormone-like activity may be low, is the urnited significance of a barely detectable response above thecontrol level. A classic criterion for determining qualitative nonidentity of 2 similar activities in biologic assay isthe appearance of nonparallel dose-response curves. Inthe absence of such a test, which requires administrationof the suspect unknown at 2 or more distinguishable doselevels 111parallel with several dose levels of the referencestandard, an important clue to the nature of the unknownis unavailable. In order to be able to inject the higher ofthe 2 dose levels required for the parallelism test, a ratherhigh concentration of parathyroid hormone-like activitymust be present in the extract, or, alternatively, the activity must be concentrated by some means.

By a mechanism not yet understood, the injection ofcysteine (0.12 M) with parathyroid hormone in the bioassayenhances the activity of the hormone about 3-fold, whichreduces the dose requirement by a like factor. However,the phenomenon is not a clear gain in the detection of mmimum amounts of hormone, since injection of cysteinealone, in comparison with uninjected controls or controlsinjected with acidified physiologic salt solution, has asmall serum calcium-raising effect which, although notusually statistically significant in individual experiments,is significant when a series of experiments are analyzedtogether (7).

Finally, it must be emphasized that even if doseresponse curves for parathyroid hormone and tumorextracts pass the test for parallelism it does not prove thatthe tumor extract contains parathyroid hormone, onlythat it contains an agent with similar activity. On theother hand, parallelism in a bioassay provides muchstronger evidence for similarity, if not identity, than aminimum effect, which is all that could be hoped for fromtumors containing a low concentration of the active agent.

The purpose of the foregoing analysis of the limitationsof biologic assay is to place in perspective the results wehave obtained from the bioassay of tumor extracts. Ourearlier assays of the hypercalcemic tumors supplied byDr. Howard and others were negative or equivocal (A. D.Kenny and P. L. Munson, unpublished observations).The positive test that we published earlier this year (9),which was based on results statistically significant onlyat the level of P < 0.05, was not regarded as strongevidence but only “consistentwith the presence of parathyroid hormone― in the tumors.

In order to make an adequate test for parathyroidhormone in the tumors in question it was necessary toturn to a method sensitive to much smaller amounts thanare required for the bioassay in rats. An immunochemicalmethod based on complement (C') fixation between para

thyroid hormone and specific rabbit anti-bovine parathyroid hormone appeared to meet this requirement (8).

The immunologic difference between human and bovineparathyroid hormone, shown by the failure of hormoneextracted from human parathyroid glands by phenol orby dilute HC1 to fix C' directly with the antibody, was aproblem. It appeared to be solved by the discovery thatextracts of human parathyroid glands made with aqueous8 M urea did fix C' directly with anti-bovine parathyroidhormone at an antibody concentration approximately 7times greater than was necessary for the homologousreaction. Furthermore, it was found that the phenol andHC1 extracts of human glands contained forms of thehormone that were antigenically similar, though notidentical to the bovine hormone, since the homologousC'-fixation reaction was inhibited by these extracts (9).The nonidentity of human and bovine parathyroid hormones has also been demonstrated immunologically byBerson et al. (3).

Nonparathyroid tumors from 6 patients with hypercalcemia were extracted (also metastases from 3 of thepatients) with urea. All 9 of the extracts fixed C' directlywith the anti-bovine parathyroid hormone. Some of thetumors were also extracted with phenol and HC1 and theextracts of all tumors thus examined inhibited the homologous C'-fixation reaction (9).

Evidence for the specificity of the direct C'-fixationreaction was provided by negative tests with urea extractsof normal tissues from the 6 tumor patients and of tumorsfrom other patients without hypercalcemia. In addition,antibody-absorption experiments indicated removal ofanti-parathyroid hormone antibody from solution by thetumor extract. Evidence for the specificity of the C'-fixation inhibition reaction was provided by the lack ofinhibition of 3 nonparathyroid immune systems byextracts that inhibited the parathyroid system (9).

Quantitative estimates of the amount of parathyroidhormone in 6 tumors and 3 metastases, based on the

TABLE 1BIOASSAYS OF TUMOR EXTRACT

a Mean value, 5 parathyroidectomized rats per group, with 1

exception. Standard errors for calcium: Experiment I, 0.66;Experiment II, 0.47—0.52;for inorganic phosphorus (Pt), 0.18.

b Cysteine (0.12 M) was added to the extract just before ad

ministration to the assay rats. The volume of cysteine solutionor tumor extract injected per rat was 2.5 ml in Experiment I and4.0 ml in Experiment II.

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1064 Cancer Research Vol. 25, August 1965

inimunoa.ssay of urea extracts, were reported to range thyroidectomized rats. When the results of the 3 assaysbetween 0.9 and 39 jig/gm, or in units, 3—135/gm. Ex- were combined, the increase in serum calcium was barelytrflCts of 3 of the tumors estimated to contain 35—45 significant statistically at. P < 0.05. If valid, the resultsunits/gm by immunoassay were hioassayed in para- would have indicated au amount. of hormone in the tumor

TABLE 2% INHIBITIONOF C' FIXATIONBY DILUTEACIDEXTRACTS

IMMUNE SYSTEM BEING INHIBITED

Egg albumin-@ Human growth. anti-egg hormone-anti

@ albumin@ growth hormoneI@I@Iti@@@@ ferritin

CMC-PTH-antiPTHU

Seph@PTH@anti@PTHbINHIBITOR

Dilution of inhibitor

000000

000000

00

9

0

27 1 3 9 27

4032153440

373010299

8260

1419

00

13

73322829

1005532

100

431426

000

08400

0000000

3300

000

0

00

02

9@ 27

000000

23456

789

10111213141516171819

20

212223

24252627282930

I@3 9 27 I@3@9 27 1 3

00000000

0000800000

@100

0 0 0 0 0 0

000

Tumor No. 1

Tumor No. 2c

Tumor No. 4d

Normal humant issues'

Parathyroidglands

LiverKidney

Tumors not associated with hy

percalcemia'53

227

10202419@89251010

0@00

I

050@0@0.23

100000

1580070

000

0000

0“CMC-PTH,

parathyroid hormone purified on carboxymethylcellulose following Sephadex G-100 (donated by Drs. J. T. Potts,Jr., and G. D. Aurbach). The C' fixation being inhibited was that obtained with 0.55—1.25@gof hormone and a dilution of antiserumof 1:500-1 :3000. No important qualitative or quantitative differences were noted in the C'-fixation curves obtained with this antigenand the highly purified hormone (Seph-PTH) used in the original study (9).

b Seph-PTH, parathyroid hormone purified on Sephadex G-50 (as described in Ref. 10). The C' fixation being inhibited was that

obtained with 0.6 @gof hormone and a dilution of antiserum of 1:500-1:3000.C Two different HC1 extracts of tile same tumor were used. The 1st dilution of extract was equivalent to 150—300 mg of tumor.

d Three different HCI extracts of the same tumor were used in this series' of experiments. The 1st dilution of extract was equiva

lent. to 100-300 mg of tumor., Extracts prepared exactly the same a.s for tumors. The 1st dilutions of the liver and kidney extracts were equivalent to >300 mg

of tissue, while the 1st dilution of the parathyroid gland extract was equivalent to only 1 mg of tissue.

I Two different tumors were extracted, a pancreatic adenocarcinoma and an adenocarcinoma of the rectosigmoid. The 1st dilut ions

of these extracts were equivalent to approximately 300 mg of tissue.

0N

S 1 3

20

18780

2128

50 236030

63 34 22

000000

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MUNSON et al.—Parathyroid Hormone in Tumors 1065

extract by bioassay consistent with the results by immunoassay. However, we did not regard the bioassay as athoroughgoing confirmation of the immunoassay results,for the reasons outlined earlier.

There ai-e 3 types of experiments that have led us torevise our conclusions (9) concerning the concentration ofparathyroid hormone-like activity iii the tumors associated with hypercalcemia.

First., additional biologic assays failed to confirm thehigh estimates by immunoassay. In these experiments,higher dose levels of the extract of a tumor were used thanin the experiment published, so that clearly positiveresults should have been obtained if the results by immunoassay had been correct. The higher doses were achievedby concentrating the urea extracts by a method similar tothat used by Aurbach for preliminary purification ofphenol extiacts of bovine parathyroid glands (1).

To test the applicability of this procedure to ui-ca cxtracts, ground, frozen bovine parathyroid glands (undissected) were extracted with aqueous 8 @rurea (20mi/gm) and the urea extract. was concentrated by precipitation of impurities with acid-acetone-NaCl, followed byprecipitation of the activity with ether. The estimatedpotency in the bioassay of a sample of the ether precipitatetaken up in 0.01 N HC1 led to a value of 110@ 1.25 @g(385units) of parathyroid hormone per gm of original tissue,which agrees well with our previous estimates of hormonecontent of undis@sect.ed bovine glands. The value obtamed by immunoassay of the ether precipitate by directC' fixation was 60 pg/gm, in fair agreement with thebioassay.

The i-esults of the 2 biologic assays of the partiallypurified urea extract of t umor are given in Table 1. Therewas no significant effect (above the cysteine conti-ol) oneither Plasma calcium or urine phosphate iii ExperimentI, and in Experiment II, although the effect on serumcalcium a@)proached statistical significance, P was > 0.05.The dose of extract given was such that there should havebeen a clearly positive effect if the tumoi- had coiitainedas much as 2—4,@g of parathyroid hormone per gin oftissue, a level lower 1han that estimated fi-om inimunoassays by diiect complement fixation of urea extracts (10jig/gm) (9). Although it has been shown that the hiologic activity of parathyroid hormone in vwo may be lowor negligible with preservation of full immunologicactivity (10), there was no reason to believe that this wasthe cause for the discrepancies between the 2 differentassay methods applied to tumor extracts.

Secondly, a preliminary experiment by R. Melick, .J. T.Potts, Jr., amid G. D. Aurbach (personal communication),using the radioimmunoassay of Berson et al. (3) to testurea extracts of hypercalcemia tumors, including 2 tumorspreviously assayed by us, indicated that there was nomore than 0.025 as much parathyroid hormone-likeactivity present as we had originally estimated by directC'-fixation.

Thirdly, it was found that when urea extracts of tumoi-sfrom 3 of the original hypercalcemic patients (Nos. 1, 2,mild 4) were incubated wit.h C' and numerous rabbit sera,immune and nonimmune, at dilutions of 1/100—1/200, areaction simulating C' fixation took place with some of theS(1FIL, although not with all of them. The l)l'esen('e or

extent of the reaction was not correlated with the antibodycontent of the serum. When tumor extracts were incubated with specific anti-parathyroid hormone in thesame experiment in which control antisera were used, theamount of C' fixed with the anti-parathyroid hormone wasusually but not invariably greater than that obtained withthe unrelated sera. It thus became apparent that a.reaction was occurring between some component of theC' system, rabbit serum, and urea ext racts of tumors fromhypercalcemic patients that was not specific for antiparathyroid hormone. Since the antigen controls wereconsistently not anti-complementary, the reaction requiredrabbit serum. We have not determined the nature of thiscomplicating reaction . However, the reaction betweenanti-parathyroid hormone and the urea extracts of tumorsdoes not merely reflect. the high protein content of t heantigen, as no such reaction has been seen between alit il)arathyroid hormone and mammydifferent human seta atlow dilution where the nonspecific lrotein content exceedsthat of the tumor extracts.

These 3 lines of evidence required us to revise o@rpreviously published estimates of parathyroid hol-mollelike material in tumors. The question could also beraised as to the existence of any such material in thetumors.

Data from biologic a.s.says, for i-easons already given,are inadequate either to support or to deny the @)I@esen(@eof parathyroid hormone iii the hypercalcemic tumors.

The radioimmunoa.ssay data of Melick, Potts, andAurbach, while provocative, also do not support thepresence of @)arathyroid hormone in the tumors in anyunequivocal mariner. These unpublished preli mi narydata, kindly communicated to us by the investigators,showed a positive reaction with urea extracts of tumors atthe borderline of sensitivity of the radioimmunoa.ssaymethod. However, the reaction was unlike that of j@rii@athyroid hormone in that the use of increased amounts ofthe urea extracts did not cause a consistent or pai-allelincrease in the extent of the reaction.

Our conclusion that a. substance immunologicallysimilar to or identical with human parathyroid hormone ispresent in hypercalcemic tumors now rests solely on newexl)eliments demonstrating the inhibitory effect of HC1extracts on C' fixation by bovine parathyroid hormuoneand anti-bovine parathyroid hormone.

Dilute acid extracts WeIC chosen for study because ofthe previous observation that. treatment of bovine i)aIathyroid hormone with acid converts the molecule froman antigen that. fixes C' directly with antibody to onethat does not. fix C' directly but still reacts with antibodyas evidenced by inhibition of C' fixation (9). For theseexperiments, a sample of the purest parathyroid hormonecurrently available, l)urified further following gel filtrationthrough Sephadex G-100 (2) by chromatography oncarboxymethylcellulose, was used as the antigen againstwhich the tumor extracts were to be tested by competitionfor antibody. This prepat-ation was kindly donated byDrs. J. T. Potts,@ and G. D. Aurbach. Similar resultswere obtained with 2 ant i-parathyroid sera produced indifferent rabbits against. parathyroid hormone purifiedby gel filtration (10). Dilute HCI extracts of 3 of theoriginal tumors (Nos. 1, 2, and 4), which had been stored

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Immune systemApproximate molecularweight of antigenAmount

ofanti@en addedto gmveUlAXImum C â€f̃ixa

lion (pg/6ml)aParathyroid

hormone8,5001.7Bovinepancreaticribonuclease13,5000.01fl-Chainhumanhemoglobin16,5005.5Human

growthhormone22,0000.03Swinepepsinogen42,0000.01Eggalbumin46,0000.05Humanhemoglobin66,0000.05Bovine

serumalbumin68,0000.04Calfskintropocollagen130,0000.1Lactic

dehydrogenase150,0000.05Horsespleenferritin460,0000.03Bacteriophage

T4 DNA1 .2 X1080.01BacteriophageT42.4 X1080.12Pneumococcus

polysaccharidebo@o@ColicineKbØ@3Dogfish

pepsinogenb

1066 Cancer Research

TABLE 3THE RELATIONSHIP OF ANTIGEN CONCENTRATIONTO MAXIMUM

C' FIXATION

The data in Table 2 on the inhibitory effect of acidextracts of tumors, although indicative of the presence ofa parathyroid hormone-like substance in the tumors, arenot adequate for quantitative estimates. It was observedthat the maximum C' fixation by pure parathyroid hormone reacting with several different rabbit anti-parathyroid hormone sera required 1.5—2.0 @gof antigen.As shown in Table 3, this exceeds, by a factor rangingbetween 6 and 180, the amount of other antigens usuallyrequired for maximum C' fixation by this technic. Theoutstanding exception, other than parathyroid hormone,with a requirement of 5.5 @gof antigen for maximumfixation, was the denatured fl-chain of human hemoglobin(5), which aggregates in aqueous media. We havepreviously published data indicating that aggregation ofparathyroid hormone occurs in neutral buffers, andsuggest that the large amount of hormone required toreach maximum C' fixation reflects, at least in part, thisproperty of the antigen in solution. Since it was knownthat the direct C'-fixing antigen was probably aggregated,but since the physical state of the acid-extracted inhibitoror the stoichiometry of the inhibition were not known, itwas not considered justifiable to estimate the hormone content of the tumors by the inhibition reaction of acid cxtracts using calculations based on stoichiometry. Additional experiments with purified human parathyroidhormone as a standard, when it becomes available, andfurther studies of acid extracts of human parathyroidglands may make semi-quantitative estimates feasible inthe future.

The limitations of the inhibition reaction, using diluteHC1 extracts, and of direct C' fixation, using urea extracts,have been emphasized in this paper. However, it isimportant to point out that direct C' fixation with parathyroid hormone, partially or highly purified from phenolextracts of bovine glands, and anti-parathyroid hormoneis a specific reaction (8) that can be used to obtain validestimates of immunologically active hormone. Parathyroid hormone was found not to react with antiseradirected against other antigens and anti-parathyroidhormone was found not to react with other linear polypeptides (A. H. Tashj ian, Jr., and L. Levine, unpublishedobservations).

ACKNOWLEDGMENTSWe are indebted to Mrs. Arlene Schaefer and Miss Elaine Col

lura for technical assistance and to Mrs. Elizabeth A. Moore forstatistical work.

REFERENCES

1. Aurbach, G. D. Isolation of Parathyroid Hormone after Extraction with Phenol. J. Biol. Chem., p54: 3179—81,1959.

2. Aurbach, G. D., and Potts, J. T., Jr. Partition of ParathyroidHormone on Sephadex G-100. Endocrinology, 75: 290—92,1964.

3. Berson, S. A., Yalow, R. S., Aurbach, G. D., and Potts, J. T.,Jr. Immunoassay of Bovine and Human Parathyroid brmone. Proc. Natl. Acad. Sci. U. S., 49: 613—17,1963.

4. Goldberg, M. F., Tashjian, A. H., Jr., Order, S. E., and Dammm, G. J. Renal Adenocarcinoma Containing a ParathyroidHormone-like Substance and Associated with Marked Hypercalcemia. Am. J. Med., 36: 805—14,1964.

5. Hill, R. J., Konigsberg, W., Guidotti, G., and Craig, L. C.

a These values are given for a dilution of antibody which will

give 70-80% maximum C' fixation.b Molecular weight unknown.

at —20°C,were prepared as previously described (9). Itwas m-easoned that if the reaction between pure bovineparathyroid hormone and its homologous antibody couldbe inhibited by HC1 extracts of tumors obtained from thehypercalcemic patients, but not by similar extracts ofnormal tissues or tumors from nonhypercalcemic patients,and if the C'-fixation inhibition reaction could be shownto be specific for the parathyroid immune system, then itwould be reasonable to conclude that the tumors containeda substance immunochemically similar to parathyroidhormone.

The results, shown iii Table 2, indicate consistentinhibition of the parathyroid hormone immune system byacid extracts of 3 tumors associated with hypercalcemia.Similar extracts of normal tissues and of tumors notassociated with hypercalcemia did not inhibit this system.Each inhibition mixture was controlled at all dilutions andany tubes showing any interference with C' activity wereexcluded. The reason for the variability in amount ofinhibition observed from one experiment to another is notclear. However, it may be due in part to differences inthe concentration of the inhibitor in different extractsprepared from the same tumor on different dates or tochanges on storage of the extracts. The lack of anysignificant inhibition of the 5 nonparathyroid immunesystems tested is evidence for the specificity of the inhibition of the parathyroid hormone immune system. Theoccasional value of 10 % or less inhibition of nonparathyroid systems shown in Table 2 is not significant.However, the unlikely possibility is not excluded thatextracts of the tumors combine with parathyroid hormoneand prevent its interaction with antibody, a result thatwould also inhibit C' fixation.

Vol. 25, August 1965

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MUNSON et al.—Parathyroid Hormone in Tumors 1067

The Structure of Human Hemoglobin. J. Biol. Chem., p37:1549—54,1962.

6. Kenny, A. D., Roth, S. I., and Castleman, B. Bio-Assay ofHuman Parathyroid Extracts. J. Clin. Endocrinol. Metab.,@4:375-80, 1964.

7. Munson, P. L. Biological Assay of Parathyroid Hormone. In:R. 0. Greep and R. V. Talmage (eds.), The Parathyroids, pp.94—113.Springfield, fli. : Charles C Thomas, Publisher, 1961.

8. Tashjian, A. H., Jr., Levine, L., and Munson, P. L. Immuno

assay of Parathyroid Hormone by Quantitative ComplementFixation. Endocrinology, 74: 244—54,1964.

9. . Immunochemical Identification of Parathyroid brmone in Nonparathyroid Neoplasms Associated with Hypercalcemia. J. Exptl. Med., 119: 467—84,1964.

10. Tashjian, A. H., Jr., Ontjes, D. A., and Munson, P. L. Alkylation and Oxidation of Methionine in Bovine Parathyroid Hormone : Effects on Hormonal Activity and Antigenicity. Biochemistry, 3: 1175—82,1964.

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1965;25:1062-1067. Cancer Res   Paul L. Munson, Armen H. Tashjian, Jr. and Lawrence Levine  Associated with HypercalcemiaEvidence for Parathyroid Hormone in Nonparathyroid Tumors

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