Use diethylenetriamine (D.T.P.A.) in assessment …Useofdiethylenetriaminepenta-acetic acid(D.T.P.A.) in the clinical assessment oftotalbodyiron stores 897 test ofstorage iron, andthis

J. clin. Path. (1967), 20, 896

Use of diethylenetriamine penta-acetic acid(D.T.P.A.) in the clinical assessment of

total body iron storesL. W. POWELL AND M. J. THOMAS

From the Medical Professorial Unit and the Department ofPathology,Royal Brisbane Hospital, Brisbane, Australia

SYNOPSIS As a quantitative test of iron storage the six-hour urinary excretion of iron was measuredafter the intravenous infusion of the iron-chelating agent diethylenetriamine penta-acetic acid(D.T.P.A.). The value of this test in determining the presence and magnitude of the total body ironstores was assessed in patients with diseases of iron storage. In patients with idiopathic haemo-chromatosis and in their first-degree relatives with increased iron stores as measured by hepaticbiopsy, the result of the D.T.P.A. test fell progressively as iron stores were reduced by venesectiontherapy. There was a significant correlation between the result of the D.T.P.A. test and the excessiron storage as measured retrospectively by the effects of venesection therapy, and also betweenthe D.T.P.A. test and the unsaturated iron-binding capacity (U.I.B.C.) in these patients.

In patients with excess iron stores due to diseases other than idiopathic haemochromatosis (e.g.,alcoholic cirrhosis, haemochromatosis associated with refractory anaemia) the D.T.P.A. testdemonstrated the presence of excess iron stores, but the results were very variable and did notcorrelate closely with the U.I.B.C. or the degree of hepatic haemosiderin deposits. Unexpectedlyhigh results for the D.T.P.A. test were found in some patients with iron-deficiency anaemia.

The demonstration that iron-chelating agentspromote increased urinary excretion of iron inpatients with excess iron stores has led to the use ofthese agents in the study of iron storage disease.Both diethylenetriamine penta-acetic acid (D.T.P.A.)and desferrioxamine have been used in simplequantitative tests in which the urinary iron excretionfollowing the injection of a known dose of chelatingagent is measured at six or 24 hours (Walsh, Perkins,Blackburn, Sanford, and Cantrill, 1963; Keberle,1964; Lloyd, Powell, and Thomas, 1964; Ploem, deWael, Verloop, and Punt, 1966; Fielding, O'Shaugh-nessy, and Brunstrom, 1966). However, both agentshave limitations when used to assess quantitativelythe degree of excess storage iron.

Desferrioxamine has more than one metabolicpathway; a variable proportion of the ferrioxamineformed in vivo is used fer haemoglobin synthesisand is not excreted in the urine (Bannerman,Callender, and Williams, 1962). This can be allowedfor if desferrioxamine is injected together with aknown dose of ferrioxamine labelled with 59Fe, and

Received for publication 21 April 1967.

the six-hour excretion of ferrioxamine and 59Femeasured (Fielding, 1965). Desferrioxamine alsochelates iron released from the breakdown ofhaem in addition to storage iron and thus highchelation values are obtained in haemolytic condi-tions and in disorders of haem metabolism (Fielding,1965; O'Shaughnessy, Brunstrom, and Fielding,1966; Smith, Studley, and Williams, 1967).D.T.P.A. is a less specific chelating agent for iron

than desferrioxamine; small quantities of othercations such as copper, magnesium, lead, and zincare chelated in addition (Tripod, 1964). However,the metabolic pathway of D.T.P.A. followingintravenous injection appears to be relatively simple.It is distributed widely in the extracellular compart-ment; 80 to 90% ofthe administered dose is recoveredunchanged in the urine in the first four hours; itdoes not participate in normal iron exchange be-tween effete red cells, reticuloendothelial cells, andtransferrin; and it appears that only tissue iron ischelated (Stevens, Rosoff, Weiner, and Spencer,1962; Cleton, Turnbull, and Finch, 1963). Theseproperties suggest that D.T.P.A. should be a valuablechelating agent for use in a simple quantitative

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Use of diethylenetriamine penta-acetic acid (D.T.P.A.) in the clinical assessment of total body iron stores 897

test of storage iron, and this has been confirmed bythe findings of Walsh et al. (1963) in patients withidiopathic haemochromatosis.A previous study (Powell, 1966a) has shown that,

in rabbits loaded with iron-dextran, the results of aquantitative test using D.T.P.A. correlated withthe total body iron excess when the test was per-formed at a constant time interval after iron loading.The present study was undertaken to assess thevalue of such a test in determining the presence andmagnitude of increased total body iron stores inpatients with various diseases of iron storage. Anindication of the state of the iron stores in thesepatients was also obtained by measurement of theserum-iron, total iron-binding capacity (T.I.B.C.),the extent of haemosiderin deposition in hepaticbiopsy specimens and bone marrow aspirates, andwhere possible, by means of repeated venesection.

METHODS

SERUM IRON AND URINARY IRON The serum-iron levelwas estimated by the method of Trinder (1956) and thetotal iron-binding capacity (T.I.B.C.) by the method ofRamsay (1957). Urinary iron was measured by a wet-ashing technique. To 5 ml. of urine was added 0-2 ml.concentrated sulphuric acid and 0 5 ml. concentratedperchloric acid. During the final stage of digestion 0 1 ml.of hydrogen peroxide (30% w/v) was added and themixture heated to dryness. After cooling, 2 ml. of waterwas added and the contents heated to boiling point andagain allowed to cool before adding 1 drop of thiogly-collic acid and 2-0 ml. of 0-2x%a, x-dipyridyl in buffer(2-0 M sodium acetate/acetic acid, pH 5 0). After mixingand standing for 10 minutes the solutions were read in aspectrophotometer with appropriate standards andblanks which had been treated in the same manner as thetest samples.

D.T.P.A. TEST (LLOYD et al., 1964) The patient's bladderwas emptied and the urine discarded. A solution con-taining 1 g. of D.T.P.A. in 500 ml. of physiological salinewas then infused intravenously over exactly one hour.All urine was collected for the six-hour period after thestart of the infusion.

Aspiration liver biopsy was performed with a Menghinineedle. Tissue was fixed in acid formalin for 48 hoursbefore processing. Sections were stained with haema-toxylin and eosin and for free iron by Perls' technique.Iron deposition was graded according to Scheuer,Williams, and Muir (1962).

CRITERIA FOR VENESECTION Weekly venesections of500 ml. were performed on those patients with cirrhosisand grade 4 iron deposition in liver biopsy specimens.In each case the presence of excess body iron stores wasconfirmed by the failure of the haematocrit or haemo-globin concentration to fall by 25% or more after sevenweekly venesections (Haskins, Stevens, Finch, and Finch,1952; Finch and Barnett, 1957). Weekly or fortnightly

venesections each of 500 ml. were then continued untilthere was evidence of iron depletion as judged by asubnormal haemoglobin concentration, a low serum-iron level and high total iron-binding capacity. Firstdegree relatives with evidence of increased iron stores inliver biopsy specimens and the D.T.P.A. test were alsosubmitted to venesection therapy as above. The otherpatients in this study did not undergo regular venesection.

RESULTS

NORMAL SUBJECTS (33) The mean values for serumiron, T.I.B.C., and urinary iron excretion in theD.T.P.A. test are shown in Table I. The serum ironwas significantly higher in males than females(P < 0 05) but no sex difference was found in theD.T.P.A. test. The 95% confidence limit for theD.T.P.A. test was 0 29 mg.

TABLE IMEAN VALUES i S.D. IN NORMAL CONTROLS

Subjects Serum Iron T.LB.C. Percentage D.T.P.A.(qg./IOO ml.) (pg./100 ml.) Saturation Test (mg.)

of T.I.B.C.

Males (14)1 125 ± 33-3 327 ± 46Females (19) 101 ± 32-3 334 ± 68Total (33) - -

'Total number in parentheses.

37 ± 9 0-16 ± 00631 ± 10 0-15 ± 0-08- 0-15 ± 0-07

IDIOPATHIC HAEMOCHROMATOSIS The details ofvenesection therapy in patients in this group aregiven in Table II. In five patients with untreatedhaemochromatosis the urinary iron excretion inthe D.T.P.A. test ranged from 41 mg. to 12-4 mg.(mean 8-6 mg.). A further nine such patients hadreceived treatment by repeated venesection buttheir serum transferrin had remained almost fullysaturated; the result of the D.T.P.A. test in thesepatients ranged from 1-2 mg. to 16-0 mg. (mean6-3 mg.). In five patients who had been treated byweekly venesection until the haemoglobin concen-tration and serum iron levels were low, the resultsof the D.T.P.A. tests were normal, ranging from0-003 mg. to 029 mg. (mean 0 18 mg.). In twopatients with idiopathic haemochromatosis andin four first-degree relatives with increased ironstores, the D.T.P.A. test was repeated at intervalsof three to six months during treatment by weeklyor twice weekly venesection (see Figures 1 and 2).In each case the urinary iron excretion in theD.T.P.A. test fell progressively as the excess storageiron was reduced by therapy. Table III shows theresult of the initial D.T.P.A. test in each subjectcompared with the total body iron stores as measuredby venesection therapy until evidence of irondepletion occurred, and in one patient (case 14), bychemical estimation of iron in tissues obtained atnecropsy. Although the number of subjects studied



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898 L. W. Powell and M. J. Thomas

TABLE IICLINICAL AND PATHOLOGICAL FINDINGS IN PATIENTS WITH DISEASES AFFECTING IRON METABOLISM

Patient Sex Age Serum Iron T.I.B.C.1 Liver Biopsy Marrow D.T.P.A. Remarks(Mg./100 ml.) (Mg./100 ml.) (grade iron)2 Aspiration Test (mg.)

(grade iron)(0-4)

Idiopathic haemochromatosis: untreatedI M 54 281

2 F

3 M

52

65

4 M 40

5 M 65

217

190

242

230

281

228

228

284

290

4 12 4 5 g. iron removed after D.T.P.A.test: venesection continuing

11t9 15 5 g. iron removed after D.T.P.A.test: venesection continuing

8-8 3-2 g. iron removed: patient thenrefused further treatment.

6-0 Total of 13-2 g. iron removed afterD.T.P.A. test

41 Died from hepatoma 2 monthslater.

4

Idiopathic haemochromatosis: incompletely treated6 F 64 254 266

M 65 264 288

M 62 - -

M 44

M 47

M 65

F 44

F 39

M 58

335

267

211

233

251

330

Idiopathic haemochromatosis: completely treated415 M 63 78

16 M 46 44

4 M 40 38(see above)12 F 44 45(see above)13 F 39 28

335

298

235

264

272

342

2 5 6 25 g. iron removed beforeD.T.P.A. test: venesection dis-continued because of anginal pain

1-2 16 5 g. iron removed beforeD.T.P.A. test: venesectioncontinuing

3 5 7 5 g. iron removed beforeD.T.P.A. test: venesectioncontinuing

9 4 7-8 g. iron removed before D.T.P.A.test and continuing

5 5 12 0 g. iron removed beforeD.T.P.A. test and continuing

5 3 > 8-0 g. iron removed beforeD.T.P.A. test: irregular attender

7-2 15-0 g. iron removed after D.T.P.A.test


16 0 Iron content of tissues at necropsy= 37-4 g. 3

4

4

4

4

356

412

430

300

296

0

0

0 003 20 g. iron removed before D.T.P.A.test.

0-2 18-5 g. iron removed beforeD.T.P.A. test


- 0-18 19-0 g. iron removed beforeD.T.P.A. test


Relatives ofpatients with haemochromatosis: completely treated'17 M 42 210 -

18 M 46 156 288

19 F 41 112 280

20 F 35 121 274

Secondary haemochromatosis21 M 8

22 F 36

23 M 9

198

399

250

412

294 294

3Fibrosis +-

2

2

4 9 11 g. iron removed after D.T.P.A.test



- 0 55 1-9 g. iron removed after D.T.P.A.test

3 4 9-6 Refractory anaemia. Approx. 15 g.Fibrosis + iron given parenterally

- 4 9-4 Sideroblastic anaemia > 50 g. irongiven parenterally. Figlu test42 mg./hr.

- 4 4-6 Thalassaemia major

'T.I.B.C. = total iron-binding capacity"Grading after Scheuer et al. (1962)"Reported in detail elsewhere (Tyrer, Powell, and Burnett, 1966)'Treatment by venesection, considered complete when patient developed a low haemoglobin concentration, low serum iron level, and high totaliron-binding capacity.

9

10

I1

12

13

14

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TABLE I -continuedCLINICAL AND PATHOLOGICAL FINDINGS IN PATIENTS WITH DISEASES AFFECTING IRON METABOLISM

Patient Sex Age Terum Iron T.L.B.C.' Liver Biopsy Warrow D.T.P.A. Remarks(j,ug./100 ml.) (Mg.1100 ml.) (grade iron)2 Aspiration Test (mg.)

(grade iron)(0-4)

Alcoholic cirrhosis with haemosiderosis24 M 51 10725 M 50 25026 M 62 21727 M 60 25528 F 57 14429 M 56 9730 M 42 104

31323334

F 43M 64M 26M 41

97122255

298364

282414307320

342290282

Alcoholic cirrhosis without haemosiderosis35 M 51 72 -36 F 48 129 416

37 M 54 72 404

Chronic alcoholism without cirrhosis3839404142

434445

M 55M 37M 53M 50M 52

M 70M 48M 45

Cryptogenic cirrhosis46 F 4447 F 4748 M 2249 F 64

Haemosiderosis50 M 6751 M 6052 M 5453 M 6554 F 6455 M 5556 M 58

Iron deficiency57 F 4558 F 2159 F 3860 F 2061 F 5262 F 8063 F 5664 F 3365 M 7866 M 4967 F 56

4010532

96

126112

92675696

24821222619695

157

443616185626322850

117

3222222

3322

00

0

264376326

322

340330

278428364312

262282256284388

298

396416520354360432370416524-

330

0101°

00

I

0-18 Wernicke's encephalopathy019 Hepatomegaly 18 cm.3-1 Serum amylase 530 Somogyi units0-39 Macrocytic anaemia0-09 -0-44 Indurated pancreas at laparotomy1-4 Abdominal pain: urinary diastase

80 Wohlgemuth units0-16 Macrocytic anaemia0.19 -0 7 Hypogammaglobulinaemia0-39 -

- 0-26 Previous haematemeses- 0-27 Wernicke's encephalopathy,

peripheral neuritis- 0-22 -

0

0000

0

0

3342

22

00000000000

0-23 Cardiomyopathy0-15 -0-28 -0-29 -0-2 Duodenal ulcer, previous

haematemesis0-15 -0-28 -0-25 -

0-32 Haematemeses0-25 Haematemeses, sister of patient 400-19 Ulcerative colitis0-15 -

6-3 Aplastic anaemia2-4 Aplastic anaemia0-98 Untreated pernicious anaemia0-2 Untreated pernicious anaemia0-1 Treated pernicious anaemia0-3 Treated polycythaemia vera0-3 Treated chronic myeloid leukaemia

0-22 Previous menorrhagia0 3 Aspirin-induced melaena0-8 Previous menorrhagia0-94 Melaena0-21 Melaena0-1 Dietary0-18 Haematemeses0-2 Previous menorrhagia0-1 Duodenal ulcer4-5 During oral iron therapy0-17 Three months after cessation of

iron therapy

Chronic pancreatitis68 F69 F70 F

366660

364

176

_ 0 0-17 -0-14 -0-5 Diabetes mellitus

I

89

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Hb. (g% )

Serum Fe and T.I.B.C.(,ug.%)

D.P.T.A. TEST (mg.)

NET IRON REMOVED (g)20 V

16 V

12 V

a

4

1964 1965

FIG. 1. Serial tests in a patient with idiopathic haemochromatosis during venesection therapy. Note the pro-gressive fall in the result of the D.T.P.A. test during therapy whilst the serum-iron and haemoglobin concentrationremained virtually unchanged, but the U.I.B.C. rose. (Interrupted lines indicate the upper limits of normal forfemales for haemoglobin concentration serum-iron and D.T.P.A tests.)

TABLE IIIRELATION BETWEEN URINARY IRON EXCRETION IN THE

D.T.P.A. TEST AND BODY IRON STORES IN FOUR PATIENTSWITH HAEMOCHROMATOSIS AND FOUR RELATIVES WITH

INCREASED IRON STORES

Patient Initial D.T.P.A. Iron Removed byTest (mg.) Venesection after

D.T.P.A. Test (g.)

412

13

1417181920

607 2(after 4 g. ironremoved)6-0(after 3 g. ironremoved)1604.90.510460 55

13 215

8-5

37.411 12 52 11.9

'Iron content of major organs of iron storage measured on tissuesobtained at necropsy (Tyrer et al., 1966).

is small the correlation is significant (r = 0984,P < 0-001).

In these patients studied serially the response toD.T.P.A. appeared to depend on the degree ofunsaturation of the iron-binding capacity of theserum, although the relationship was not a simplelinear one (Fig. 3).

Figure 2 demonstrates an observation whichwas made on three patients when treatment byvenesection had been suspended because the serumiron, the haemoglobin concentration, and the resultof the D.T.P.A. test had all fallen to subnormallevels. After a further two to three months the serumiron and the result of the D.T.P.A. test had returnedto abnormally high levels, and a further 1-5 g. ofiron could be removed by venesection before thelevels fell again to normal or subnormal values.

SECONDARY HAEMOCHROMATOSIS (TABLE 11) In thethree patients with secondary haemochromatosisand chronic anaemia (one each with refractory

900

1s

12

9

6

400

300

200

100

.8

6

4

2

L.0000JUNE1 963



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Use of diethylenetriamine penta-acetic acid (D. T.P.A.) in the clinical assessment of total body iron stores 901

A.W.

1512g Hb gI.163

Serum Fe and t11.C.(lpg.1.j400300200100

0

6 .T.P.A. TEST lmgJ6

2O m_ m _ 1 t_ 1 1 -

CUMULATIVE TOTAL (g.116 OF IRON REMOVED

12 b.12j-~~~~~ ~ ~ ~~~~~~~~~~~~~~~~~-

~~~~~~~~~~~~~~~~~1

FED JUL OC MAR1964

FIG. 2. Serial tests in a patient with idiopathic haemo-chromatosis. Venesection therapy was withheld in March1965 for eight weeks. The serum iron and result of theD.T.P.A. test returned to abnormally high levels and afurther 1.5 g. of iron was then removed.(Interrupted lines indicate the upper limits of normal formales for haemoglobin concentration, serum-iron andD.T.P.A. tests.)

anaemia, sideroblastic anaemia, and thalassaemia)the serum iron levels were elevated with increasedsaturation of the T.I.B.C. and there was excesshaemosiderin in bone marrow aspirates. In thesepatients the increase in urinary iron excretion in theD.T.P.A. test (9-6, 9-4, and 4-6 mg. respectively)was similar to that in patients with untreatedhaemochromatosis although the total iron storesprobably varied widely (see discussion).

ALCOHOLIC CIRRHOSIS (TABLE ii) Fourteen patientswith alcoholic cirrhosis were studied. In 11 of thesestainable iron was present in the liver biopsy butthe degree of siderosis (maximum grade 3, meangrade 2) was never as gross as in the cases of haemo-chromatosis (grade 4). None had been treated byrepeated venesection. This group was distinguishedfrom idiopathic haemochromatosis on the basis ofthe degree of iron excess and by the fact that familystudies had failed to reveal excess iron storage in afirst-degree relative. In six of these 11 patients theurinary iron excretion in the D.T.P.A. test wasincreased. However, the mean excretion (0-93 mg.)

was much lower than that of patients with haemo-chromatosis and the D.T.P.A. test did not showthe correlation with the degree of hepatic irondeposition which was found in patients with un-treated idiopathic haemochromatosis (Powell, 1965).In the three patients with alcoholic cirrhosis withouthaemosiderosis the result of the D.T.P.A. test fellwithin the normal range.

CHRONIC ALCOHOLISM WITHOUT CIRRHOSIS (TABLE II)In all eight patients with chronic alcoholism theD.T.P.A. test was normal. The serum iron levelwas reduced in two and normal in four otherswhilst the T.I.B.C. was within the normal rangein all six tested. Liver biopsy showed no stainableiron in five, and in the remaining three mild haemo-siderosis (grade 1) which is normal (Powell, 1966b).

CRYPTOGENIC CIRRHOSIS (TABLE ii) The results ofthe D.T.P.A. test and the serum iron were withinthe normal range in all four patients in this group.In each case liver biopsy revealed an inactivecirrhosis with no stainable iron present.

HAEMOSIDEROSIS (TABLE II) Seven patients werestudied in this group. All suffered from disorders oferythropoiesis commonly associated with excessiveiron storage and in five patients excess haemosiderindeposition was demonstrated in bone marrowaspirates. In three of the seven patients the D.T.P.A.test was abnormal although the result showed noconstant relation to the degree of haemosiderosis inbone marrow aspirates or to the serum iron level.There was a weakly significant correlation betweenthe D.T.P.A. test and the U.I.B.C. in these patients(r = -0-88, P < 0-05). Liver biopsy was not per-formed on any patient in this group.

IRON DEFICIENCY (TABLE II) The D.T.P.A. test gaveunexpectedly high results in three of the nine patientswith untreated iron-deficiency anaemia. Only one ofthe three patients had suffered from menorrhagiaand in this patient there was no vaginal blood lossat the time the test was performed. A further patienttested during oral iron therapy excreted 4-5 mg.iron in six hours in the D.T.P.A. test and in an-other patient tested three months after cessation oftherapy the result was normal.

CHRONIC PANCREATITIS (TABLE II) In one of thethree patients with chronic pancreatitis the D.T.P.A.test was increased to 0-5 mg. It is of interest thatthis patient had diabetes mellitus but there wereno other clinical stigmata of haemochromatosis.She refused further investigation.



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8

6F \ x

3l-.

CO 4La3-.

QC

2 x

100 200 300

UNSATURATED IRON BINDING CAPACITY(jg

DISCUSSION

This clinical study has confirmed the practical valueof the D.T.P.A. test in demonstrating the presenceof increased total body iron stores and in facilitatingthe quantitative assessment of excess body iron inpatients with idiopathic haemochromatosis. In thiscondition the results showed a gradation from thehigh levels obtained in untreated patients down tothe normal levels in those patients who had beentreated until the serum iron and haemoglobinconcentrations were below normal. These resultsagree closely with the findings of Walsh et al. (1963).In four patients with haemochromatosis and fourimmediate relatives, there was a significant correla-tion between the urinary iron excretion in theD.T.P.A. test and the amount of storage iron asmeasured by repeated venesection or, in one case,by chemical estimation of iron after necropsy(Table III). When serial D.T.P.A. tests were per-formed in six subjects during the period of treatmentthe fall in urinary iron excretion was not accom-panied by a fall in serum iron or haemoglobin leveluntil virtually all the storage iron had been removed(Figs. 1 and 2). Thus, during the period of treatmentby venesection, the D.T.P.A. test gives a betterindication of the amount of storage iron presentthan do the serum-iron and the haemoglobin

FIG. 3. Relation between D.T.P.A.test and unsaturated iron-bindingcapacity in the case illustrated inFigure 2. The correlation coefficientbetween X and log Y is r = --0-927(P < 0 01).

400per100 ml.)

concentration. In these six patients studied seriallythe response to D.T.P.A. showed a significantnegative correlation with the degree of unsaturationof the iron-binding capacity although the relation-ship was not a simple linear one (Fig. 3). Similarfindings were observed with desferrioxamine byWalsh, Mass, Smith, and Lange (1965). However,this observation may not necessarily mean that theamount of iron chelated by D.T.P.A. is determinedby the amount of circulating unsaturated transferrin.Walsh et al. (1963) showed that, in patients withtreated haemochromatosis in whom the transferrinhad become resaturated, the urinary excretion ofiron provoked by D.T.P.A. was only slightly in-creased. In other words the serum iron was elevatedand the T.I.B.C. fully saturated when there was arelatively small excess of body iron. Further increasein iron stores did not alter these values, but wasshown by a further increase in urinary iron excretionin the D.T.P.A. test. In our patient A.W. (Fig. 2),resaturation of transferrin was associated with asubstantial increase in response to D.T.P.A. In thepresent study, the result of the D.T.P.A. test inpatients with alcoholic cirrhosis and haemosiderosisshowed no constant relation to the serum iron ortotal iron-binding capacity.The observation illustrated in Fig. 2 shows that

not all storage iron has necessarily been removed by

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venesection therapy when evidence of iron deficiencyappears. During the period in which venesectionswere withheld the iron absorbed from the gut wouldprobably not have exceeded 5 mg. per day (Haskinset al., 1952). This is equivalent to 280 mg. over theeight-week period whereas 1,500 mg. was sub-sequently removed by venesection. It is probablethat during this eight-week period the remainingiron, previously non-mobilizable and non-chelatable,was converted to a form which was both chelatableand readily mobilized. In case 12 (Fig. 1) a liverbiopsy specimen obtained when the serum iron andresult of the D.T.P.A. test had fallen to low levelsshowed some haemosiderin deposits still present.

Accurate measurement of body iron stores inpatients with haemochromatosis and their relativesis difficult and there is debate about the best methodcurrently used (Balcerzak, Westerman, Lee, andDoyle, 1966). Measurement of iron removed bymeans of repeated venesection is probably the mostaccurate method as almost all tissue iron is availablefor haemoglobin synthesis (Haskins et al., 1952).However, this method provides only retrospectiveinformation. Liver biopsy is a simple, practical andrelatively safe means of examining tissue iron storesby histochemical staining and also permits theassessment of liver damage. The amount of ironwhich stains histochemically, however, is not alwaysdirectly proportional to the total body iron stores.In patients with untreated haemochromatosis theamount of iron which can be removed by a com-plete course of venesection may vary from 15 to 40 g.despite similar appearances (grade 4) of liverbiopsy specimens (unpublished observations). Inone patient with grade 3 iron deposition in the liver(Table II, patient 17) a total of 11 g. of iron wasremoved by venesection, and in three other patientswith grade 2 iron deposition the amount of ironremoved by venesection was 2-5, 21, and 19 mg.respectively. Patients with alcoholic cirrhosis mayhave moderate haemosiderosis of the liver (grades2 to 3) with only a slight increase in chelatable bodyiron (Powell, 1965; Walsh et al., 1965) and normal orslightly increased amounts (1 0 to 2 5 g.) of ironmobilizable by venesections (Conrad, Berman, andCrosby, 1962; Williams, Williams, Scheuer, Pitcher,Loiseau, and Sherlock, 1967). Quantitative testsof iron storage using iron-chelating agents canprovide useful additional information about thedegree of iron excess. The present study has demon-strated a relationship between the urinary ironexcretion in the D.T.P.A. test and the amount ofstorage iron removed subsequently by venesections.Thus, the D.T.P.A. test is of value in the quantitativeassessment of excess iron storage in patients withidiopathic haemochromatosis, but liver biopsy is

necessary to determine the degree of tissue damage,the presence of cirrhosis, and the distribution ofstorage iron within the liver.

In patients with diseases of iron storage otherthan idiopathic haemochromatosis (e.g., alcoholiccirrhosis, secondary haemochromatosis associatedwith chronic anaemias such as sideroblastic anaemiaand thalassaemia) the D.T.P.A. test demonstratedthe presence of excess iron stores but the resultshowed no constant relation to the degree of ironexcess as estimated by the serum iron, the T.l.B.C.,and liver and marrow haemosiderin deposits. Insuch conditions the disorder of iron storage iscomplicated by ineffective erythropoiesis, increasediron absorption, and recent parenteral iron ad-ministration, each of which may affect ironchelation. However, as none of these patients wassubjected to venesection therapy, firm conclusionscannot be drawn about the relationship betweenthe D.T.P.A. test and body iron stores in thesepatients.The results of the D.T.P.A. test in patients with

iron deficiency were rather unexpected. Two of thethree patients in whom the results were abovenormal had suffered from gastrointestinal bleedingbut it is unlikely that contamination of urine withblood would account for the results obtained.Normal or high levels of urinary iron excretionafter the administration of chelating agents havealso been found by other workers in iron deficiency(Hwang and Brown, 1964). A possible explanationfor this phenomenon is that in such patients a smallamount of storage iron remained but that it wasbeing rapidly mobilized and therefore was mostlyin chelatable form.

We would like to thank Professor J. H. Tyrer and Pro-fessor A. W. Pound for their constant support andencouragement and for access to facilities in the Depart-ment of Medicine and the Pathology Department,Royal Brisbane Hospital, respectively. We are alsograteful to Dr. W. Inglis and Geigy Pharmaceuticals(Australasia) Ltd. for supplies of D.T.P.A. and forcontinued support.

REFERENCES

Balcerzak, S. P., Westerman, M. P., Lee, R. E., and Doyle, A. P.(1966). Amer. J. Med., 40, 857.

Bannerman, R. M., Callender, S. T., and Williams, D. L. (1962).Brit. med. J., 2, 1573.

Cleton, F., Turnbull, A., and Finch, C. A. (1963). J. clin. Invest.,42, 327.

Conrad, M. E., Jr., Berman, A., and Crosby, W. H. (1962). Gastro-enterology, 43, 385.

Fielding, J. (1965). J. clin. Path., 18, 88.--, O'Shaughnessy, M. C., and Brunstrom G. (1966). Ibid., 19, 159.Finch, S. C., and Barnett, R. N. (1957). New Engi. J. Med., 256,

884.Haskins, D., Stevens, A. R., Jr., Finch, S., and Finch, C. A. (1952).

J. clin. Invest., 31, 543.Hwang, Y. F., and Brown, E. B. (1964). Arch. intern. Med., 114, 741.



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O'Shaughnessy, M. C., Brunstrom, G. M., and Fielding, J. (1966). J.clin. Path., 19, 364.

Ploem, J. E., de Wael, J., Verloop, M. C., and Punt, K. (1966). Brit.J. Haemat., 12, 396.

Powell, L. W. (1965). Quart. J. Med., 34, 427.(1966a). Rev. med.-chir. Mal. Foie, 41, 271.

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Smith, P. M., Studley, F., and Williams, R. (1967). Lancet, 1, 133.Stevens, E., Rosoff, B., Weiner, M., and Spencer, H. (1962). Proc.

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The September 1967 IssueTH4E SEPTEMBER 1967 ISSUE CONTAINS TH4E FOLLOWING PAPERS

Obituary H. A. MagnusResults with radioisotopic assay of serum B12 usingserum binding agent D. M. MATTHEWS, R. GUNASEGA-RAM, and J. C. LINNELL

Vitamin B12 absorption studies: effect of parenteral non-radioactive vitamin B12 on serum level of 57Co vitaminB12 LILIAN HARWOOD and J. FORSHAW

Estimation of intrinsic factor and detection of intrinsicfactor antibodies using non-radioactive cyanocobalaminand microbiological assay G. H. SPRAY

A comparison of two methods for the immunoassay ofintrinsic factor K. D. BARDHAN, A. G. WANGEL, andSHEILA T. CALLENDER

Incidence and pathogenesis of megaloblastic erythro-poiesis in multiple myeloma A. V. HOFFBRAND, J. R.HOBBS, S. KREMENCHUZKY, and D. L. MOLLIN

Adenosine diphosphate and the measurement of plateletadhesiveness A. L. BLOOM and A. J. DAVIES

Postoperative changes in platelet adhesiveness P. N.BENNETT

Retention of platelets by glass bead filters w. R. PITNEYand MARY POTTER

Autofluorescence of bone tissues A. I. D. PRENTICE

Developments in immunofluorescence: the need forstandardization G. D. JOHNSON, E. H. BEUTNER, and E. J.HOLBOROW

Cesar: cervical smear analyser and reader. A newapproach to evaluating cells in cytological preparationsI. M. P. DAWSON, C. P. HEANLEY, A. C. HEBER-PERCY, andJ. K. TYLKO

Synthetic progestogen-oestrogen therapy and uterinechanges J. G. AZZOPARDI and I. ZAYID

Malignant granular cell myoblastoma D. H. MACKENZIEVariation in subcapsular liver structure and its signi-ficance in the interpretation of wedge biopsies M.PETRELLI and P. J. SCHEUER

Neutrophil alkaline phosphatase in pregnancy R. W.BEAL, W. M. F. READ, and P. A. TURVEY

Evaluation of microscopic methods in the diagnosis ofidiopathic steatorrhoea B. K. PATNAIKTreatment of chronic urinary tract infections withgentamicin A. A. LINDBERG, H. BUCHT, and L. 0. KAL-LINGS

Use of a solid reagent in the triphenyl tetrazolium chloridetest for bacteriuria N. A. SIMMONS and J. D. WILLIAMSAutomatic serial diluting: an instrument for use inbacteriological laboratories R. E. TROTMAN

Automatic platelet counting with the Coulter particlecounter R. E. DAVIS and R. E. GREEN

Precision in a clinical chemistry laboratory J. D.ACLAND and S. LIPTON

Technical methodSimple ultrafiltration apparatus w. H. C. WALKERLetter to the EditorBook reviewsInternational standard for haemoglobinometry s. M.LEWIS

Training of post-mortem room technicians J. F. ILEGGIE

Copies are still available and may be obtained from the PUBLISHING MANAGER

BRITISH MEDICAL ASSOCIATION, TAVISTOCK SQUARE, w.c.l., price 18s. 6D.

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