phosphatase activity (lagothrixwith alkaline phosphatase levels perhaps somewhat raised (i have no...

J. clin. Path. (1962), 15, 99

Phosphatase activity in the limb bones of monkeys(Lagothrix humboldti) with hyperparathyroidism

GRACE M. JEFFREE

From the University of Bristol Pathology Research Laboratory'

SYNOPSIS The paper reports a study of the distribution of phosphatases in the femora of threespecimens of Humboldt's woolly monkey (Lagothrix humboldti) suffering from chronic hyper-parathyroidism. Bone structure ranged from the apparently normal to extreme osteitis fibrosa.Most marked changes were found in the distribution of alkaline phosphatase, which reached atleast 10 times the normal levels in the bone of the second monkey in the series, dropping to levelsstill well above normal in that of the most severely affected animal. Very high concentrations werefound in the deeper layers of hypertrophied growth cartilage and in the osteoblasts lining poorlycalcified trabeculae, and high concentrations in the fibre bone of the third animal. Lack of minerali-zation and the development of osteitis fibrosa are thus associated with a marked increase in alkalinephosphatase activity. Osteoclasts reacted strongly for acid phosphatase but were negative foralkaline phosphatase. Acid phosphatase levels were comparatively high in fibre bone, but overalllevels ranged from 1/20 to less than 1/100 those of alkaline phosphatase. Some slow staining foracid phosphatase probably represents residual activity at acidpH of the markedly increased alkalinephosphatase. There may be some association between a failure of mineralization and the presenceof acid phosphatase in osteoclasts and osteoid.The aetiology of the monkeys' condition is discussed. It seems likely that the parathyroid hyper-

trophy and rachitic changes were caused by low blood calcium dependent on a low calcium dietand lack of vitamin D, in which the requirements of New World monkeys are reputedly high.

In 1959-60, the Bristol Bone Tumour Registerreceived, through Mr. J. A. Dall, the bodies of fivemonkeys from Paignton Zoo. Four animals had asevere bone disorder, and the parathyroid glandsappeared markedly enlarged in all five. The animalsare listed in Table I. All but the green monkey wereSouth American species. The three specimens ofHumboldt's woolly monkey formed a series inwhich the condition of the bones ranged fromapparently normal to extreme deformity. A studyof the distribution of phosphatases in their femoraforms the subject of this paper.A brief resume of the history of simian bone

disease given by Ruch (1959) is relevant to the con-dition of these animals. Workers early in this centuryreported a condition in captive monkeys char-acterized by thickened skull bones, enlarged jawbones, and tumour-like swellings of the nasal bones,identifying it with the human tropical diseasegoundou, a rare tertiary lesion of yaws. It was laterReceived for publication 30 September 1961.'18 Guinea Street, Bristol, 1.

realized that this facial deformation was only onemanifestation of a generalized bone disorder ofcaptivity, which was identified by Corson White(1922b) with Paget's osteitis deformans of bone,though, even while making this identification, hedrew attention to the similarity to osteitis fibrosacystica and also to the calcium-deficient diet of theaffected animals. Later workers suggested that thecondition resembled von Recklinghausen's disease.Ruch suggests that the disease is one of dietary

insufficiency: 'A factor probably more powerfulthan words was that the pseudo-goundou-Paget'sdisease entity disappeared from collections ofcaptive monkeys and apes as a result of improveddiets introduced widely at this time.' Again,'Throughout the period of 1895-1935 almost no onegave serious consideration to the possibility thatsome of the processes which result in rickets andosteomalacia in man manifest themselves in monkeysin an unusual fashion. This oversight was repeatedeven though many of the animals simultaneouslyshowed obvious signs of rickets or osteomalacia.

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TABLE ICONDITION IN MONKEYS FROM PAIGNTON

Parathyroids Bone Condition

(mg.) (mg./kg.)

A BTR/ 1277Humboldt's woolly monkey, adolescentB. BTR/1254Humboldt's woolly monkey, adolescentC BTR/1215Humboldt's woolly monkey, adolescent

BTR/1278Capuchin, adult

BTR/ 1280Green monkey, adult

M 2-698 29 10-7 Bones well calcified and apparently normal

M 4 536 44 9 7 Bones soft and flexible, several fractures, hypertrophiedgrowth cartilages, mandible could be cut with knife

F 1-927 91 47-2 Bones very soft; advanced and extensive osteitis fibrosacystica, with large and microfractures. Hypertrophiedgrowth cartilages, mandible easily cut with knife

F 1-474 38 25-1 Bones generally softened, callus formation from pastincomplete fractures, and healing fracture of femur,mandible readily cut with knife

M 6-397 30 4-7 Bones slender and somewhat softened, fractures L.humerus and radius, mandible soft, but too hard to cut

Yet the pathologic picture in some animals was

sufficiently different from rickets and osteomalaciaof man to prompt the thought that the monkeydiffers from man in some important way in osseousfunction, mineral metabolism, dietary requirements,or endocrine make-up. There is always the possibilitythat, in the decay of interest in simian bone patho-logy, an interesting and important aspect of physio-logy and pathology has been lost sight of.'Although osseous lesions resembling Paget's

osteitis deformans of mammals other than manhave been described, closer investigation has in ourhands shown characteristic differences, and thereare good reasons to suggest that osteitis deformansis a skeletal disease entirely restricted to the humanrace. Osteitis fibrosa in many mammals affects firstthe jaw and skull bones. 'Rubber-jaw' of dogs is a

late manifestation ofhyperparathyroidism secondaryto leptospiral nephritis (Platt, 1951); 'big-head' ofequines is an extra-European disease associated withcomparative excess of phosphorus in the diet, andmanifesting acid urine with hyperphosphaturia,slightly high serum P, and slightly low serum Ca(Hutyra, Marek, and Manninger, 1946). The prob-ability that in the monkeys from Paignton Zoo thehyperparathyroid state was secondary to rickets willbe considered in the discussion.

MATERIALS AND METHODS

The three specimens of Humboldt's woolly monkey wereall adolescent, with epiphyses still open. They have beendesignated A, B, and C in order of increasing severity ofbone changes. Brief notes of necropsies performed byDr. C. H. G. Price are given below.

A (BTR/1277) A male, weighing 2-698 kg., which diedsuddenly on 15 January 1960. The body was received on17 January and necropsy performed on 20 January.The animal appeared relatively healthy, though thin, andthe cause of death was not found. The lymph nodes were

slightly enlarged; the kidneys were gritty to the knife,suggesting some metastatic calcification; otherwise theviscera appeared normal. The bones were firm and wellcalcified, without deformities or fractures. Two para-thyroid glands were found, weighing 14 and 15 mg.,making a total of 29 mg., or 10-7 mg. per kg. body weight.This was the mate of the animal C (BRT/1215), whichdied on 15 February 1959 when it also was thoughtlikely to die from the same condition. By July 1959,however, it had fully recovered. A sample of blood serumwas obtained on 15 October 1959 which was analysed asfollows:-Calcium, 121 mg./100 ml.; phosphorus, 9-4mg./100 ml.; alkaline phosphatase, 296 King-Armstrongunits/100 ml.; acid phosphatase, 23-6 K-A. units!100 ml.The serum was markedly haemolysed, which mightexplain the high value obtained for acid phosphatase,since there is a high concentration of this enzyme inerythrocytes. It would not, however, explain the very highlevel of alkaline phosphatase.

B (BTR/1254) A male, weighing 4 536 kg., was killedon 16 July 1959 and necropsied on 17 July. This animalshowed generalized lymphadenopathy. The liver andkidneys were congested, but otherwise normal. Bonestended to be soft and flexible, particularly the distalbones; there were fractures in the right femur and tibia,the left tibia and fibula. The mandibles were very softand easily cut with a knife through the horizontal ramus.Four parathyroid glands were found, weighing 7, 13, 8,and 16 mg., making a total of 44 mg. or 9-7 mg. per kg.body weight. The glands were of very loose texture,slightly acinar, and composed of transitional, chief, andclear cells. No adenomata were found.

c (BTR/1215) A female, weighing 1-927 kg., whichdied on 15 February 1959 and was necropsied on 17February. The body was generally in good condition,though rather poorly nourished. Lymph nodes wereenlarged; the kidneys showed marked cloudy swellingand widespread mild tubular nephritis, with cellularproliferation in the glomeruli. There were extensiveareas of necrosis with abscess formation in the liver. Thebones showed well-advanced and very extensive osteitisfibrosa cystica, with both large and microfractures andmuch soft-tissue swelling (e.g., of periosteum), with

100

Animal

PARATHYROIDS

Sex

AND BONE

Weight(kg.)



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Phosphatase activity in the limb bones of monkeys (Lagothrix humboldti) with hyperparathyroidism 101

wispy new bone laid down in large amounts but poorlymineralized. Several growth cartilages were distortedand deformed, with poorly developed primary spongiosa.The marrow was generally hyperplastic, rather congestedand with areas of thrombosis; fatty marrow was generallyreplaced by swollen myxoid fibrous tissue. The mandiblewas very soft, and easily cut transversely with a knife.Two parathyroids were found, with a total weight of 91mg., giving a ratio of 47 mg. per kg. body weight. Theglands were nodular, acinar, with chief and clear cellsand transitional cells. No adenomata were found.

QUANT1TATIVE PHOSPHATASE ESTIMATIONS The bones ofthe right limbs were dissected out and stored in the freez-ing chamber of a domestic refrigerator before examina-tion. Quantitative estimations were carried out as in aprevious study of normal rabbit bones (Jeffree, 1959).Bones were cleaned, weighed, and drawn to scale, thencut longitudinally, and half or a quarter of each bonedivided into portions on an anatomical basis; eachportion was finely chopped and weighed. Separateportions were well ground in a glass or porcelain mortarwith a little acid-washed silver sand and a measuredquantity of distilled water, to give known dilutions ofbone (the sand, being insoluble, can be neglected in thecalculation). Initial dilutions varied from 1 in 10 to 1 in50, according to the amount of bone available. Thesuspensions were left in the refrigerator overnight, thenshaken and centrifuged; the supematant fluid was usedfor phosphatase estimations, higher dilutions of theoriginal extract being made where necessary for esti-mations of alkaline phosphatase. Para-nitrophenyl phos-phate was used as substrate, according to the modificationof the method of Bessey, Lowry, and Brock (1946)described by Jeffree (1959). Distribution maps are com-parable with those in the latter paper.

HISTOLOGICAL DEMONSTRATION OF PHOSPHATASES Por-tions of bone removed from the freezing chamber werefixed overnight in the body of the refrigerator in absoluteethanol, 10% formol saline (approximately 3-5% form-aldehyde), or 5% formol saline buffered atpH 7-2. Thesewere then dehydrated and embedded in paraffin wax, or

double-embedded in low-viscosity nitrocellulose andparaffin according to the method of Ball (1957), whosetechnique for cutting undecalcified sections was usedfor most of the slides illustrated, though with the softbones of monkeys B and C it was possible to cut sectionsof single-embedded tissue on a Cambridge rockingmicrotome. The results of the two methods may be com-pared in Fig. 3. Fig. 3B shows material which was

double-embedded and cut on a base sledge microtome,with Sellotape support for the section, by the method ofBall, whereas the softer bone of Fig. 3C was single-embedded and cut on a Cambridge rocking microtomewithout support; even in this very soft tissue the cartilagecells have been displaced in the process of sectioning.A few sections were stained for phosphatase according

to the methods of Gomori (1946, 1950) as quoted byPearse (1960) but it was found preferable to use themethod of Burstone (1958a and b), which avoids falsepositive reactions in nuclei,cartilage matrix, and undecal-

cified bone, and shows the relationship of phosphatasestaining to mineralized structures which take the hae-malum counterstain.

RESULTS

QUANTITATIVE ESTIMATIONS OF ALKALINE PHOSPHATASEFig. 1 shows the distribution of alkaline phosphatasein the right femora of the three monkeys and in thatof a 5-month-old rabbit. The femur of A is roughlycomparable to this rabbit bone, but shows somewhathigher overall values and not such a marked con-centration of the enzyme below the epiphyseal plates.This appears to be a normal or nearly normal bone,with alkaline phosphatase levels perhaps somewhatraised (I have no means of assessing the speciesdifferences). The bones of the other two animalsshow marked divergence from the normal. Levelsof alkaline phosphatase in B are enormously high,being about 10 times those in A, as will be seen bycomparison with the inset figure, in which phos-phatase values have been shown at one tenth theiractual level, giving a picture very similar to A; thefemur of B, in fact, shows a roughly normal dis-tribution of alkaline phosphatase at 10 times thenormal level. The distribution in C is markedlydifferent. There are raised values (though not as highas in B) throughout the bone, except in the periosteumand the fractured head, where very low values arefound. The most marked deviation from normal is inthe shaft, where the thickened, fibrous bone shows aconcentration of the enzyme very much greater thanin a normal compact shaft wall.

QUANTITATIVE ESTIMATIONS OF ACID PHOSPHATASEFig. 2 shows the distribution of formaldehyde-stableacid phosphatase. Estimations were made in thepresence of 0 5% formaldehyde in order to minimizethe contribution of erythrocyte acid phosphataseand to give values comparable with those of theprevious study of rabbit bones (Jeffree, 1959). Itshould be noted that, in view of the smaller concen-trations of this enzyme, shadings in this diagramrepresent only one tenth the activity shown bysimilar shadings for alkaline phosphatase. The valuesof acid phosphatase in the femur of A, like those ofalkaline phosphatase, are somewhat higher than inthat of the 5-month-old rabbit, except below thegrowth cartilages, where they are lowered ratherthan raised. The femur of B shows similar valuesbut a slightly different distribution, with raised con-centrations below epiphyseal and articular cartilagesand in the endosteum. Acid phosphatase in the femurof C is generally lower than in the other two animalswith a more even distribution throughout the bone,except for the fractured head which shows extremelylow levels of the enzyme.



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102 Grace M. Jeffree

BTR 1277 'i

... S~~~~~~~~~~~~~~~~TR1215

00

gM p-NITROPHENYL PHOSPHATE HYDROLYSED/ HR /GM BONE AT 37°C AND pH 10-3

UNDER 10 10-20 20-40 40-60 60-80 80-100 100-200200-400400-600600-800 800-10000VER1,000

FRI(. I Alkaliie phosphatase of m?onkey .femtora.

CARLI IT C LAf%kUe



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RABBIT 5 MONTHS

A cBT R 1277

FIG. 2. Formaldehyde-stable acid phosphatase of monkey femora.

B dBTR 1254

C ?BTR 1215

pM p-NITROPHENYL PHOSPHATE HYDROLYSED/ HR/GM BONE AT 37°C AND pH 4-9

UNDER I 1-2 2-4 4- 6 6-8 8-10 10-20 20-40 40-60



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A 49Zr~~~li

FIG. 3. Alkaline phosphatase in epiphyseal plates offemora.Incubations at room temperature andpH 83.

Substrate Naphthol AS-TR phosphate.Diazotate Fast Red TR salt.

At

FIG. 3A

A

.,.'

FIG. 3A. Four and a half hours' incubation. x 120.

FIG. 3A and 3B. Fixed coldformol saline buJffred at pH7-2; double-embedded and cut on base sledge microtome,with Sellotape support for sections.

FIG. 3B. One and a half hours' incubation. x 60.

FIG. 3B

-:.Fifv.w

, 'N .: jt 4i.je e.

*5

FIG. 3C. Fixed cold,formol saline; paraffin embedded andcut on Cambridge rocking microtome.

"E' ;'..,

'% s

FIG. 3C. Three hours' incubation. x 60.

FIG. 3C

104

.. .. A.

ll



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HISTOCHEMICAL STAINING OF ALKALINE PHOSPHATASEThis enzyme is demonstrated in four main sites:I In the deeper layers of cartilage cells of both arti-cular and epiphyseal cartilage; 2 in the oesteoblastson trabecular surfaces; 3 in the inner layer of theperiosteum; and 4 in the fibrous tissue between tra-beculae in the affected bones of B and especially C.Table II gives comparisons between the three

femora. The growth cartilage in both B and C ismarkedly hypertrophied, and irregular in depth,with a tendency to separate into tongues as in typicalrickets (Fig. 3). In A there is also some irregularityin depth, but the cartilage is much narrower andshows a well-mineralized zone of provisional calci-fication. In the metaphysis, wide osteoid zones areapparent in B and C (Fig. 4). Osteoclasts are inevidence only in B, and here only in moderatenumbers. Fibre bone is a noteworthy feature of Cand a few fibres are found between the trabeculae ofB but A shows well-formed cortical bone whichappears to be fully mineralized.The very high levels of alkaline phosphatase found

quantitatively in B are reflected to some extent in theshorter periods of staining required for the bones ofthis animal.

HISTOCHEMICAL STAINING FOR ACID PHOSPHATASEWith some exceptions, staining these bones forphosphatase at pH 5 to 5 4 gives a picture verysimilar to that obtained at pH 8-3, suggesting thatthis staining may be largely due to residual activityof alkaline phosphatase at the acid pH. Allen andHunter (1960) found, on zymograms of epididymal

enzymes, two bands reactive for phosphatase at pH6, of which one represented acid phosphatase per seand the other the residual activity of alkalinephosphatase. Bone sections stained for alkalinephosphatase were incubated at room temperature,but for acid phosphatase at 37°C. and generally forconsiderably longer periods, so that like intensity ofstaining by no means represents comparable enzymeactivity. Under these conditions the residual activityof alkaline phosphatase, which quantitatively maybe 100 times as active as the acid phosphatase, is afactor to be considered. However, there are somedifferences in the distribution of staining at acid pHwhich are likely to represent the activity of acidphosphatase proper.

I Articular and epiphyseal cartilage In sectionsincubated for a moderate length of time, acidphosphatase is demonstrated in and around the cellsin the deeper layers of the columns, with a distri-bution similar to that of alkaline phosphatase(Fig. SB, 1) but in some sections incubated overlonger periods, staining is seen throughout the depthof the epiphyseal cartilage in both cells and matrix(Fig. 5B, 2). It is possible that this weak activityrepresents diffusion of the enzyme during the longperiod of storage in the refrigerator before fixation.

2 Trabeculae The presence of acid phosphatasein osteoblasts on the trabecular surfaces (Fig. 6B)is in contradistinction to the findings of Burstone(1958c) but may well be explained by the very highvalues of alkaline phosphatase in these bones andthe long periods of staining at pH 5 4, permitting aresidual reaction from this enzyme. Staining in the

,LE IIDISTRIBUTION OF ALKALINE PHOSPHATASE IN MONKEY FEMORA

al A Animal B Anin

Epiphyseal cartilage

Trabeculae of metaphysis

Short columns of cells stainingweakly for alkaline phosphatase inlower levels. Stronger staininground degenerate cells of well-mineralized zone of provisionalcalcificationFig. 3A

Wide well-calcified trabeculaewith narrow osteoid seamsbordered by narrow layer ofalkaline phosphatase-positiveosteoblastsOsteoclasts not in evidenceFig. 4A

Cartilage markedly hypertrophiedand irregular in depth, tending toseparate into tongues. Strongstaining for alkaline phosphataseto a depth of 20 to 30 of lowercells, very little mineralizationFig. 3B

Narrow trabeculae with wideosteoid seams covered by a layerof strongly alkaline phosphatase-positive osteoblasts (Fig. 4B)A fair number of osteoclasts, whichare negative for alkaline phos-phatase (Fig. 4B0)

Cartilage markedly hypertrophiedwith tendency to split into tongues.Alkaline phosphatase stainingthrough a deep zone of cells,moderate degree of mineralizationFig. 3C

Trabeculae poorly formed, partlymineralized, with wide seams ofosteoid sometimes on one side onlySome surfaces lined by cells whichare positive for alkaline phosphataseOsteoclasts not seen Fig. 4C

Subperiosteal region

Fibre bone

In all three animals the inner layer of the periosteum shows a zone of variable width of alkaline phosphatase-positive cells. The cells are elongated and appear similar to those of fibre bone Figs. 4A, 4B, and 4C

May be represented by an increased A few alkaline phosphatase-width of subperiosteal tissue in positive fibres are found betweensome places Fig. 4A the trabeculae Fig. 4B

An extensive network of fibres whichare positive for alkaline phosphataseconnects osteoid seams of trabeculaeand islets of osteoid Fig. 4C

Region Anim mal C



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X Periostell

4~~~~~~~~~4i

FIG. 4A. One and a half hours' incubation. x 60. FIG. 4B. Three quarters ofan hour's incubation. x 60.

F1GS.4A, 4B0, and 4C. Fixed coldformol saline buffered atpH 7-2; double-embedded and cut on base sledge microtome,with Sellotape support for sections.

FIG. 4B. Fixed coldformol saline; paraffin embedded and cut on Cambridge rocking microtome.

Periosteum A j' i

Osteo-

FIG. 4C. Twenty-four hours' incubation. x 60. FIG. 4BO. One and a half hours' incubation. x 227.

FIG. 4. Alkaline phosphatase in metaphyses. Incubations at room temperature and pH 8-3.Substrate Naphthol AS-TR phosphate. Diazotate Fast red TR salt.

106



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t. t,t I:;..

C *r,.a X _ '.,. ,*fl*.A_'*~4~v/& ,~ > C ,.....t

FIG. 5B. 1. Four hours' incubation. x 60.

4% .4~~~~~~~~~~~~......

th-~ ~

HG.5B.2 Eigtee hous'incbaion x60

FIG. 5. Acid phosphatase in epiphyseal plate offemur. Bone fixed in cold formol saline, buffered at pH 7-2: double-embedded and cut on base sledge microtome, with Sellotape support for sections. Incubations at 37°C. andpH 5.4.Subtrate Naphthol AS-TR phosphate. Diazotate Fast Bordeaux OL salt.

Periosteum

Osteo-clasts

>.Kt4j¾t sw

.

it.

HG. 6B. Two hours' incubation at pH 5-4. x 60. FIG. 6C. Twenty-four hours' incubation at pH 5 0. x 60.

FIG. 6B. Fixed in cold formol saline, buffered at pH 7-2: double-embedded and cut on base sledge microtome, withSellotape support for sections. Incubation at 37°C. andpH 5 4.

FIG. 6C. Fixed in cold ethanol; paraffin embedded and cut on Cambridge rocking microtome. Incubation at 37°C.andpH 5 4.

Substrate Naphthol AS-TR phosphate. Diazotate Fast Bordeaux OL salt.

FIG. 6. Acid phosphatase in metaphyses.



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osteoclasts, however, indicates the presence of acidphosphatase proper, since these cells show noreaction for alkaline phosphatase.

3 Subperiosteal region Staining here may beentirely due to a residual reaction of the alkalinephosphatase (Fig. 6).4 Fibre bone The fibre bone (Fig. 6c) of animal

C stains strongly for acid phosphatase on longincubation and the staining extends into the osteoidseams of the skimpy trabeculae and the islets ofosteoid. Since this distribution is somewhat differentfrom that of alkaline phosphatase, it may representa real association between the presence of acidphosphatase and a failure of mineralization.

DISCUSSION

These three monkeys form a series with bonestructure ranging from the apparently normal con-dition in A to extreme osteitis fibrosa in C. Thereare changes suggesting rickets in B and C and theparathyroids of all three animals are greatly enlargedby human standards and by comparison with themeagre data available for monkeys.Gilmour and Martin (1937) give the mean weights

of histologically normal parathyroid glands as120-8 mg. for men and 139-7 mg. for women.Assuming, after Brody (1945), a mean body weightof 70 kg. for men and 60 kg. for women, the normalhuman ratios will be 1-72 mg. and 2-33 mg. per kg.body weight. Gilmour and Martin's highest valuein a 'normal' human was 388 mg. of parathyroids ina stout man; if, on a conservative estimate, weassume his weight to have been 80 kg., the ratio inthis extreme instance was 4 85 mg. per kg. bodyweight, or just half the lowest value in this series ofmonkeys.The only figures available for monkeys are those

given by Baker (1942) for parathyroid volumes in amacaque, Macaca mulatta (the rhesus monkey). Hisratios for normal animals are 0-276 c.mm. per kg.body weight for males and 0 344 c.mm. for females,measuring the external parathyroids only. Accord-ing to Arndt (1923), the external parathyroids in apescorrespond to the inferior glands in man, which areslightly heavier than the superior pair, and internalglands may be present or absent in different in-dividuals of the same species. It is thus a generousestimate to assume the total parathyroid tissue inthese animals to be double that of the externalglands. If, following Gilmour and Martin (1937),we take the specific gravity of the parathyroids asI 1, we can convert Baker's figures for the macaqueto approximate ratios of total parathyroid weight tobody weight on the following basis:--

Males 0-276 x 2 x I -1=0 608 mg. per kg. body weightFemales 0 344 x 2 x l-1=0-756 mg. per kg. body weight

These are less than half the normal human ratios,making it improbable that, even in a different speciesof monkey, the normal ratios would be grosslyabove those for man.

Parathyroid hypertrophy may thus safely beassumed in all three animals, though it is mostextreme in C, the animal with the most severelyaffected bones, with a ratio of 47-2 mg. per kg. bodyweight. A similar hypertrophy was found in theparathyroids of the other two monkeys fromPaignton, whose bones are not included in thisstudy. These were both adult animals. One was afemale Capuchin (Cebus), also a South Americanmonkey, which had softened bones with evidence ofpast incomplete fractures, and a healing pathologicalfracture in the left femur. The mandible wasmarkedly softened and cut fairly readily with aknife through the horizontal ramus. There werefour parathyroids with a total weight of 38 mg. inan animal weighing 1-474 kg., giving a ratio of 25 1mg. per kg. body weight.The other animal was an Old World monkey, a

male green monkey (Cercopithecus), weighing 6 397kg., with less bone disease than any except animal Aof this series. The bones were somewhat softenedand deformed and there was an incomplete fractureof the lower shaft of the left humerus, but it was notpossible to cut the horizontal ramus of the mandiblecompletely with a knife. Only two parathyroidswere found, after an extensive search, with a com-bined weight of 30 mg., giving a ratio of 4-7 mg. perkg. body weight, the lowest in the series, and indeedwithin Gilmour and Martin's extreme limit for thenormal human, though well above the humanmean and still further above that for monkeys.

Parathyroid hypertrophy is thus present in atleast four, and probably in all five animals, and thebone changes in the green monkey and the Capuchin,with some softening of all bones, but most notablyof the mandible, are suggestive of hyperparathy-roidism. In the series of three woolly monkeys, themandible was found to be very soft in both B and C,but normal in A, in which no bone lesions werefound. The bones of animals B and C showedchanges similar to rickets, with typically hyper-trophied and irregular epiphyseal cartilages andwide osteoid seams in the metaphysis, and in Cthere was extreme osteitis fibrosa.The hyperparathyroidism may well be secondary

to the rachitic condition, but the cause of this isstill somewhat uncertain. The condition was atfirst thought to be a renal rickets consequent upona kidney infection, like the disease of dogs known

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as 'rubber jaw', in which hyperparathyroidismfollows phosphate retention by kidneys damaged byleptospiral infection (Platt, 1951). However, in viewof the mild kidney damage, even in the most severelyaffected animal C, this aetiology seems improbablefor the monkey lesion. A more likely explanation isa low calcium rickets, dependent on an absolute orrelative calcium deficiency in the diet, or on lack ofvitamin D, or a combination of both causes. Para-thyroid hypertrophy may have been initiated by thelow blood calcium.Corson White (1922a) described osteomalacia in

New World monkeys on a diet deficient in bothcalcium and vitamins and stated that this diseasewas rare in Old World macaques. He also recordedthree New World monkeys with so-called 'osteitisdeformans', which from his description was almostcertainly osteitis fibrosa (Corson White, 1922b). Thefood was similar in both instances-two applesand/or two bananas, six small sweet potatoes, and alump of boiled rice the size of an egg-and CorsonWhite himself had no doubt that calcium and vitamindeficiency was the underlying cause of both dis-orders. He stated that the diet could be renderedadequate for rats by the addition of leafy vegetablesand fresh milk and that one at least of his animalswith 'osteitis deformans' showed a marked avidityfor lime, and seemed more comfortable when treatedwith a salt mixture containing calcium phosphateand calcium lactate, though no other change wasnoted in its condition. The choice of calciumphosphate was perhaps unfortunate, as this would dolittle to redress a calcium/phosphorus imbalance, butit is also possible that, as in man, once parathyroidhypertrophy was established, the condition wouldbe autonomous, even after removal of the originalcause. Fiennes (1961) has found that osteo-dystrophy in monkeys rarely responds to vitamin Dtherapy; but the probability is that any treatment,short of parathyroidectomy, would be ineffectiveonce the condition was fully established.Table III gives the calcium and phosphorus

content and the Ca/P ratio of a number of foods.All those listed by Corson White have a low Ca/Pratio and lack vitamin D, which would have assistedin the utilization of the available calcium. Sherman(1941) states that 'observations upon several specieshave indicated that during rapid growth and calcifi-cation it is well that the food as a whole should havea Ca/P ratio somewhere between I and 2. Whenadult maintenance only is involved, the calciumrequirement is lower, both absolutely and relativelyto the phosphorus requirement'.The food of the monkeys at Paignton Zoo during

1959-60 is stated to have been a mixture of rootvegetables and fruit, mincemeat, egg, and milk.

TABLE IIICALCIUM AND PHOSPHORUS IN FOODS'

Food

ApplesBananasLean beefBread, whiteBread, whole wheatBrussels sproutsCabbage (headed)Cabbage (loose leaf or greens)CarrotsMaizeDatesEggs (whole)GrapefruitGrapesKaleLettuceCow's milkMuttonOatmealOnionsOranges (or orange juice)ParsleyParsnipsPearsPeanutsPeas, dryPeas, freshPotatoesRadishesRaisinsRice, entireRice, whiteSweet potatoTomatoes (seeds included)TumipTurnip topsWatercress

Ca P Ca/P(mg./100 g.) (mg./100 g.) Ratio

7 11 0-648 28 0-2913 204 0-06(5) (10) (0-5)(6) (37) (0-16)25 105 0-2445 28 1-61

429 72 5-9642 40 1-0529 281 0-1072 60 1-2058 224 0-2617 18 0-9517 21 0-81

181 67 2-7054 31 1*74118 93 1-2715 208 0-0781 365 0-2232 44 0-7325 19 1-32193 84 2-3057 80 0-7113 16 0-8166 392 0-1773 397 0-1822 122 0-1813 53 0-2537 31 1-1955 110 0-568 336 0-209 92 0-10

33 52 0-641 1 27 0-4151 32 1*59

254 58 4-38168 41 4-10

'Data from Sherman (1941)

This sounds a reasonably balanced diet, but maystill have had a low Ca/P ratio if the proportions ofmeat and egg, which are both very high in phosphoruscontent, were large compared with that of milk,whereas a sufficiency of milk would supply bothcalcium and vitamin D. The amount of sunlightreceived by the aninrals while in the zoo is uncertain;although many of the monkey cages have outsideruns, some get sunlight only through glass, and it isnot certain which cages these animals occupied.However, it is probable that much of the damageantedated the animals' arrival at the zoo. In animalC at least, which was purchased from dealers only10 weeks before its death in February 1959, thecondition of the bones and the extreme hypertrophyof the parathyroids suggest a much longer history.Its mate, animal A, was at this time thought to besuffering from a similar condition, but had appar-ently made a full recovery by July 1959 and at death,in January 1960, its bones were well calcified andappeared normal, despite the parathyroid hyper-trophy. Animal B was purchased from dealers in



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Grace M. Jeifree

TABLE IVSERUM ANALYSIS AND SIZE OF PARATHYROIDS ESTIMATED IN OCTOBER 1959

Ca P Alkaline Phosphatase Acid Phosphatase Parathyroids(mg./100 ml.) (mg./100 ml.) (K-A. units/lOOml.) (K-A. units/lOOml.) (mg. per kg. body

weight)

Animal

Woolly monkey A (adolescent)

Green monkey (adult)Capuchin (adult)

12-1

11-117-0

9-4 296

6-6 328- 310

23-6

6-631-4

10-7 (at death Jan.1960)4-7

25-1

June 1958 and survived at the zoo till July 1959when it was killed on account of its poor condition.No blood analyses were made for animals B and

C, but blood was obtained from animalA in October1959, and also from the green monkey and Capuchinwhich died at this time. Analyses are shown inTable IV. In all three animals the level of alkalinephosphatase is extremely high and this is no doubtassociated with the bone condition; but little reliancecan be placed on the raised figures for acid phos-phatase, as the sera were markedly haemolysed andhence contaminated with erythrocyte acid phos-phatase. Serum calcium in A is slightly but notmarkedly high; serum phosphorus is definitelyraised. In the green monkey also serum phosphorusis raised, but serum calcium is normal. In theCapuchin, serum calcium is markedly raised, butunfortunately serum phosphorus was not estimatedowing to insufficiency of serum. These figures arecompatible with the hypothesis that the initial causeof the condition was a low calcium intake due tohypovitaminosis D or a Ca/P imbalance in the diet,leading to raised blood phosphorus and initiallylowered blood calcium. Parathyroid hypertrophy,initiated by the lowered blood calcium, wouldrestore this to normal, whilst the blood phosphorusremained high. As will be seen from Table IV, thelevel of serum calcium increases with increasingparathyroid hypertrophy.

It is probable that some at least of these monkeyswere kept in cages lacking direct sunlight, and hencesuffered from avitaminosis D. Ruch (1959) mentionsthe reputedly high requirements of New Worldmonkeys in vitamin D. Cowdry and Scott (1936)found two specimens of a New World monkey(Cebusfatuellus) unaffected by large doses of vitaminD which produced kidney damage and calcificationof the arteries in macaques. Woolly monkeys innature are predominantly if not purely fruit-eaters,though the Capuchins also eat insects (Beddard,1902). Vitamin D requirements must normally bemet principally by synthesis with the aid of sunlight;it may be that these monkeys do not utilize vitaminD in food as readily as the Old World species. Itwas the opinion of a keeper at Bristol Zoo thatnatural sunlight is of far greater importance in the

prevention of bone disease in these animals thaneither vitamin supplements or artificial ultra violet.A lack of sunlight, either in the hands of dealers orin covered cages in the zoo, may possibly be thegreatest single factor in the aetiology of this con-dition.

Harrison and Fraser (1960a) record experimentson calcium and vitamin D deficiency in rats whichare pertinent to this discussion. A diet deficient incalcium, but with adequate vitamin D, led to osteo-porosis, without formation of osteoid; serum calciumand phosphorus were low, but alkaline phosphataselevels were not affected. On diets deficient in bothcalcium and vitamin D wide osteoid seams werefound in the bones though, probably because theserats failed to grow, rachitic changes at the epiphyseswere not clearly shown. Serum calcium levels werestill lower than in the rats lacking only calcium;serum phosphate and alkaline phosphatase wereconsiderably raised. Osteoclastic activity in thebones was not prominent in either condition.

In a subsequent paper (Harrison and Fraser,1960b) the effect of calcium deficiency on the para-thyroids was studied. After three months the para-thyroids of calcium-deficient rats were found to begrossly hypertrophied and between two and threetimes the size of those from control rats of the sameage, with significantly fewer nuclei per unit area.Unfortunately, the effect on the parathyroids of thediet deficient in both calcium and vitamin D wasnot investigated.Assuming a similar physiological response in the

monkey, the condition of the bones in animals B andC suggests both calcium and vitamin D deficiency.Serum analysis, for the three monkeys in which itwas made, agrees with Harrison and Fraser's findingsfor calcium- and vitamin D-deficient rats in respectof phosphorus and phosphatase levels. The normalor raised serum calcium in the monkeys may beexplained by the parathyroid hypertrophy.

In animal A the structure of the bones appearednormal despite the enlarged parathyroids, and itseems probable that the dietary deficiencies whichinitiated the hypertrophy had been made good bythe time of the animal's death. Phosphatase dis-tribution in these bones was not very different from

110



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that in the young rabbit, though the somewhatgreater concentrations in the metaphysis, particularlyof alkaline phosphatase, may be related to the para-thyroid hypertrophy. In B the level of alkalinephosphatase was extremely high throughout thefemur, which accords with the clinical experience ofhigh serum alkaline phosphatase in osteitis fibrosaand rickets, and indeed, according to Albright andReifenstein (1948), in any condition where matrix isbeing laid down in excess. It is noteworthy that, inan electrophoretic analysis of extracts from thebones of animals B and C performed by Miss S. F.Greene of the Royal Free Hospital, the alkalinephosphatase migrated as a band in the beta-globulinregion corresponding with that shown by the alkalinephosphatase of normal human serum, supportingthe assumption that the serum enzyme is in the mainderived from that of bone. The extremely high levelsof alkaline phosphatase in B were associated withthinning of the trabeculae and a marked insufficiencyof mineralization. In C, where the whole bone had alargely fibrous structure, the values were still wellabove those of normal bone, but this may be partlydue to the greater cellularity of this fibre bone.The hypertrophied epiphyseal cartilage of B and

C reacted strongly for alkaline phosphatase, sug-gesting that this is only one factor in mineralization,though there appears to have been more calcificationin the subepiphyseal region than elsewhere in thesebones, wide osteoid seams appearing lower downthe metaphysis in both animals, along with muchfibre bone in C. These fibres again, and the osteo-blasts covering poorly mineralized trabeculae,reacted strongly for alkaline phosphatase; failure ofmineralization is associated with excess rather thanlack of this enzyme.The presence of acid phosphatase in high con-

centration in osteoclasts has already been observedby a number of workers (Gomori, 1941; Changus,1957; Pepler, 1958; Schajowicz and Cabrini, 1958;Burstone, 1958c). This, together with the localincrease in acidity found around these cells byCretin (1951), may be significant in relation to theirrole in bone resorption. It is open to speculationwhether the acid phosphatase found in fibre boneand in osteoid seams and islets of osteoid maybear some relation to the failure of calcification,even in the presence of concentrations of alkalinephosphatase of the order of 100 times as great.

What local differences in pH, or in concentrationsof activators or inhibitors, may affect the activitiesof the two enzymes, are at present unknown.

This work was supported by a grant from the BritishEmpire Cancer Campaign. Dr. C. H. G. Price has givenme a great deal of help and advice, particularly on theclinical and histological aspects of the subject. We areindebted to Mr. J. A. Dall for the bodies of the monkeys,and to Mr. L. W. Cahill, the Superintendent of PaigntonZoological and Botanical Gardens, for his help over therecords of their history. I wish to thank Mr. J. E. Hancockfor the colour photographs of slides, and Miss S. F.Greene for electrophoretic analysis of bone extracts. Iam indebted to Imperial Chemical Industries, Ltd., andFarbwerke Hoechst Ag. for samples of dyestuffs used instaining.

REFERENCES

Albright, F., and Reifenstein, E. C. Jr. (1948). The ParathyroidGlands and Metabolic Bone Disease, p. 6. Williams and Wilkins,Baltimore.

Allen, J. M., and Hunter, R. L. (1960). J. Histochem. Cytochem., 8,50.

Arndt, H. J. (1923). Z. Anat. Entwick. Gesch., 68, 514.Baker, B. L. (1942). Anat. Rec., 83, 47.Ball, J. (1957). J. clin. Path., 10, 281.Beddard, F. E. (1902). Mammalia. The Cambridge Natural History,

p. 557. Macmillan, London and New York.Bessey, 0. A., Lowry, 0. H., and Brock, M. J. (1946). J. biol. Chem.,

164, 321.Brody, S. (1945). Bioenergetics and Growth, p. 388. Reinhold Publish-

ing Corporation. New York.Burstone, M. S. (1958a). J. nat. Cancer Inst., 20, 601.

(1958b). Ibid., 21, 523.(1958c). J. Histochem. Cytochem., 7, 39.

Changus, G. W. (1957). Cancer, 10, 1157.Cowdry, E. V., and Scott, G. H. (1936). Arch. Path. (Chicago), 22, 1.Cretin, A. (1951). Presse mdd., 59, 1240.Fiennes, R. N. (1961). Personal communication.Gilmour, J. R., and Martin, W. J. (1937). J. Path. Bact., 44, 431.Gomori, G. (1941). Arch. Path. (Chicago), 32, 189.

(1946). Amer. J. clin. Path., 16, 347.(1950). Stain Technol., 25, 81.

Harrison, M., and Fraser, R. (1960a). J. Endocr., 21, 197.- , (1960b). Ibid., 21, 207.Hutyra, F., Marek, J., and Manninger, R. (1946). Special Pathology

and Therapeutics of the Diseases of Domestic Animals, 5thEnglish edition, ed. J. Russell Grieg, Vol. Ill, p. 225. BailliereTindall & Cox, London.

Jeffree, G. M. (1959). J. Bone Jt Surg., 41B, 401.Pearse, A. G. E. (1960). Histochemistry, Theoretical and Applied,

2nd ed., pp. 868, 874, 881, 882. Churchill, London.Pepler, W. J. (1958). J. Path. Bact., 76, 505.Platt, H. (1951). J. comp. Path., 61, 188.Ruch, T. C. (1959). Diseases of Laboratory Primates, pp. 28, 472-500.

Saunders, Philadelphia and London.Schajowicz, F., and Cabrini, R. L. (1958). Science, 127, 1447.Sherman, H. C. (1941). Chemistry of Food and Nutrition, 6th ed.,

pp. 265, 562-565. Macmillan, New York.White, E. P. Corson (1922a). Arch. intern. Med., 30, 620.- (1922b). Ibid., 30, 790.



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