INTERNATIONAL JOIIRNAL OF LEPROSY^

Volume 61, Number 4Printed in the t'.S..I.

Altered Calcium-Binding Ability of Plasma Proteins as theCause of Hypocalcemia in Lepromatous Leprosy'

Maria C. Vidal, Oscar A. Bottasso, Amira Lehrer, andRodolfo C. Puche2

Lepromatous Leprosy (LL) is character-ized by an uncontrolled bacillary growth to-gether with multiple and widespread le-sions. The Lepromatous form of the diseaseis important not only for the maintenanccof the source of infection in the communitybut also as a cause of disabilities and a va-riety of sequelae as well as having systemicrcpercussions. In fact, LL is known to pro-duce renal and hepatic involvement alongwith several biochemical alterations such asabnormal plasma leveis of hormones, en-zymes and minerais.

Low circulating calcium leveis have oftenbeen found in LL (4 . 12 . 14 . 20 . 22 1 . 23 ) but littleattention has been paid to the causes thatmay account for this abnormality. The pres-ent study was undertaken to determine theorigin of the hypocalcemia found in LL pa-tients.

PATIENTS AND METHODSIn a first approach, plasma calcium ( 25 ),

albumin ( 5 ) and total proteins (') concen-trations were measured in plasma samplesfrom 34 I,L patients and from 36 age- andsex-matched, healthy, dose contacts. Thepatient group consisted of 24 men and 10women with a mean age of 55 ± 5.6 (S.D.)years. All of them were outpatients peri-odically attending the Leprosy Clinic fortreatment and control. Twenty-three pa-tients had active disease and seven of themhad experienced type 2 reactions in the last6 months. Six patients were newly diag-

' Rcceivcd for publication on 20 April 1993; ac-cepted for publication on 24 Mav 1993.

M. C. Vidal, M.S., Ph.D., Investigator; R. C. Puche,M.S., Ph.D., Director, Laboratorio de Biologia Osea,Facultad de Medicina, Sante Fe 3100, 2000 Rosario,Argentina. O. A. Bottasso, M.D., Ph.D., Investigator,Division Inmunologia; A. Lehrer, M.D., Servicio deDermatologia, Hospital Provincial de Rosario, Rosa-rio, Argentina.

Reprint requests to Dr. Puche.

nosed untreated cases; the rest of the pa-tients were being treated with the followingtherapeutic regimens: dapsone (DDS) 100mg daily (N = 16), rifampin (RMP) 600 mgplus DDS 100 mg daily (N = 9), and mul-tidrug therapy (MDT; DDS 100 mg/daily,clofazimine 50 mg daily, and RMP 600 mg+ clofazimine 300 mg monthly, supervised(N = 3)). The mean duration of treatmentwas 13.2 ± 7.8 years. The patients had sta-ble disease at the time of evaluation andwere free of concomitant pathological dis-orders. Plasma samples were obtained at theCentral Laboratory of the Hospital Provin-cial de Rosario and were drawn on occasionof routine laboratory screening.

In a second step, a smaller group of con-senting individuais, 10 LL patients and 10household contacts, volunteered to partic-ipate in an investigation dealing with theintestinal absorption of calcium. The LLgroup was composed of seven men and threewomen, mean age 48 ± 11 years, who wereunder treatment with MDT (9) or DDS (1).The project was approved by the EthicsCommittee of the Facultad de CienciasMédicas, Universidad Nacional de Rosario.

After an overnight fast, blood was drawnwith heparinized syringes; pH, p0,, pCO,were determined with a radiometer. Plasmawas saved to measure calcium ( 25 ), total pro-teins ('), albumin ( 5 ), alkaline phosphataseand its bone isoenzyme ( 18 ), tartrate-resis-tant acid phosphatase ( 2 ), creatinine ( 3 ) and1,25-dihydroxyvitamin D, (`'). Ionized cal-cium was measured with a flow-throughspecific electrode (Orion Biomedical SS-20).

Urinary calcium ( 25 ), creatinine ( 3 ) andhydroxyproline (") were measured on 24-hr samples. The calculation of creatinineclearances was performed by the standardprocedure.

Intestinal absorption ofcalcium was mea-sured as indicated elsewhere (l 9 ). The resultsare expressed as percentage of test dose of

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61, 4^Vidal, et al.: Iltpocalcenria in LL^ 587

THE TABLE. Plasma and urinara' components and physiological rntctions in LL subjectsand their household contacts (controls).

LL patients Controls

Calcemia, total, mg % 8.8 ± 0.1 (34)' 10.9 ± 0.3 (36)Calcemia, ionic, mg % 4.5 ± 0.2 (12) 4.2 ± 0.12 (12)Total proteins, g % 7.2 ± 0.1 (34)'' 6.6 ± 0.13 (36)Albumin, g % 4.3 ± 0.1 (34)° 3.4 ± 0.08 (36)13lood p11 7.37 ± 0.05 (10) 7.41 ±0.05(10)pCO,, rum Hg 37.0 ± 2.0 (10) 39.0 ± 5.0 (10)Plasma bicarbonate, mEq/L 21.0 ± 2.0 (10) 24.6 ± 2.0 (10)Alkaline phosphatase

Total, IU/L 124 ± 23 (10) 120 ± 25 (10)13one isoenzymc, IU/L 76 ± 12 (10) 70 ± 8 (10)

Acid phosphatasc, tartrateresistant, UGG % 4.1 ± 0.7 (10) 5.0 ± 0.9 (10)

Urinary hydroxyproline, mg/d 25.0 ± 5.0 (10) 23.4 ± 10 (10)1,25-dihydroxyvitamin D. ng/L 28.8 ± 7.9 (10) 33.0 ± 6.0 (10)Intestinal Ca absorption,

of test dose 41.8 ± 3.8 (10) 46.2 ± 11 (10)Estimated Ca intake, g/d 0.32 ± 0.08 (10) 0.44 ± 0.08 (10)Estimated Ca balance, mg/d +21 ± 45 (10) -14 ±30 (10)Creatinine clearance, ml/min 129 ± 14 (15) 120 ± 6 (15)

lndicates significant difference at the 0.05 levei.Indicates significant difference at the 0.01 levei.lndicates significant difference at the 0.001 levei.

calcium. Calcium intake was cstimatcd bymeans of a questionnaire, recalling the in-take during the last 7 days. Daily intake ofnutrients was calculated with the aid of foodcomposition tables. Calcium balances werecstimatcd by the formula:

(Ca intake x % absorbed) = urinary Ca.

The dissociation of calcium proteinatecomplexes as a function of pH was inves-tigated, adjusting the [H f] in the range 4 to120 nM (pH 6.9 to 8.4) of two pools of sera(control and LL) with 1 M acctic acid. Theprotein-unbound calcium fraction was sep-arated by ultrafiltration (") and measuredby atomic absorption spectrophotometry( 25 ). The concentration of ultrafilterable cal-cium was expressed as percentage of totalplasma calcium. The LL pool employed had7.4 g/dL of total proteins, 5.3 g/dL of al-bumin, and 8.3 mg/dL of calcium; that ofcontacts had 7.1, 3.8 and 9.8, respectively.

Student's t test and regression analysiswere employed for assessment of the data( 24 ).

RESULTSThe findings obtained in the first part of

the study (The Table) confirmed the pres-ence of hypocalcemia in LL patients con-

current with significantly higher plasmaproteins and albumin concentrations. Fur-ther study of a subset of 10 LL patients andtheir household contacts revealed, however,that plasma ionized calcium concentrationand blood pH, pCO, and bicarbonate werewithin normal values.

Investigation of food intake revealed alarge variance of the data which was attrib-uted to the wide range of social and culturalbackgrounds of LL subjects and their house-hold contacts. As expected, comparison offood intakes between the two groups yieldedno statistical differences. Carbohydrates in-take ranged from 157 g/d to 483 g/d, pro-teins from 32 g/d to 167 g/d and fat from15 g/d to 166 g/d. Food intake averaged2200 kcal/d with a range 985-3620 kcal/d.Only two patients and one contact wouldquality as undernourished according to theirresponses to the questionnaire. On the av-erage, the intake of basic nutrients of LLpatients and contacts (carbohydrates, pro-teins and fat) expressed as percentages oftotal kcalories/d were 51%, 16% and 33%,respectively, quite dose to some dietarygoals (22 ) (58%, 12% and 30%, respectively).

To address the question of whether or notmalabsorption of calcium was related to hy-pocalcemia, the plasma levels of 1,25-dihy-

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FIG. 1. A = Relationship bctwcen plasma calcium and albumin concentrations in thc plasma of 36 contacts(r = 0.503, p < 0.05). B = Rclationship between plasma calcium and albumin concentrations in thc plasma of34 LL patients (r = 0.031, p > 0.05).

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droxyvitamin D1 and calcium absorptionwere measured. As shown in The Table,similar values were obtained from bothgroups and, therefore, chronic malabsorp-

Ultraflitereble calcium 1% of calcemia)

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Flc. 2. Rclationship between ultrafilterable plasmacalcium (expressed as percentage of total calcium) andhydrogcn ion concentrations of plasma pools of LLpatients and their contacts (see text for further details).

tion and/or a disturbance of vitamin D, me-tabolism was ruled out. In agreement, Ku-mar, et al. ( 10 ) have reported normalstructure and function of the bowel of LLpatients.

In spite of their limited daily calcium in-take, all subjects investigated were deemedto be in calcium balance. In agreement withthe latter but not with hypocalcemia, theurinary excretion of hydroxyproline, theplasma concentration of alkaline phospha-tase (total and the bone isoenzyme), andtartrate-resistant acid phosphatase (markersof bone turnover, osteoblast activity andosteoclast function, respectively) were foundto be within normal values. The latterstrongly suggests normal parathyroid status.

As observed by other investigators ( 16) asignificant, positive relationship (r = 0.503,p < 0.05) could be established between cal-cem ia as a function of albuminemia (Fig.1A). Data from LL patients, however,showed no significant trend (r = 0.031, p >0.05) (Fig. 1B).

Since the overall information obtainedsuggested an abnormal relationship be-tween calcium and serum proteins, an ex-periment was devised to assess the com-petition between [Ca++] and [H+] towardthe protein binding of the former (Fig. 2).

100

90

80

70

60

61, 4^Vidal, et al.: Hypocalceinia in LL^ 589

At normal pH values, the plasma proteinsof LL patients bound a smaller proportionof total calcium than did the plasma pro-teins of the contacts. The LL patients arethus in cquilibrium with a normal concen-tration of ionized calcium in their plasma.

DISCUSSIONIn analyzing the potential causes leading

to hypocalcemia, and in agreement with Raoand Saha (2 '), undernutrition does not ap-pear as a likely explanation because of thefact that the intake of nutrients by the LLpatients were quite dose to some dietarygoals ( 22 ).

Similarly, osteomalacia was discarded asa plausible cause for LL hypocalcemia be-cause of the normal plasma levels of 1,25-dihydroxyvitamin D, and normal calciumabsorption. Bone lesions in leprosy char-acteristically are resorptive in nature (' s ) and,therefore, hyper-and not hypocalcemiawould have been expected. When investi-gating the osteoclastic resorption in maxil-lary alveolar bone in LL patients, Marksand Sabramaniam (") observed the pres-ence of large numbers of Mycobacteriumleprae Glose to osteoclastic lesions, and theyadvanced the hypothesis of accelerated boneresorption by the release of some productfrom the neighboring bacteria.

A number of studies have been reportedin which hypocalcemia was demonstratedassociated with both low ( 8 . 9 ) as well as high(' and our study) plasma protein levels. Thesize of the plasma protein compartment thencannot be invoked to explain the hypocal-cemia of LL patients.

Rao and Saha ('-') have suggested im-paired end-organ responsiveness to para-thyroid hormone or increased levels of cal-citonin as likely causes of LL hypocalcemia.The findings reported in this paper do notsupport these hypotheses.

The overall information afforded by ourpresent data indicates that the hypocalce-mia of LL patients is the consequence of thealtered ability of plasma proteins to bindcalcium. Quantitative Ca-binding experi-ments with purified plasma proteins are cur-rently in progress in our laboratory to gainfurther insight into the equilibrium [Ca-Proteinate [Ca -1- 1 + [proteins° - ] in theplasma of LL patients that leads to a normalconcentration of ionized calcium and, in

turn, a normal parathyroid status as indi-cated by several markers.

SUMMARYThis paper reports a study performed on

10 lepromatous leprosy outpatients and onthe same number of age- and sex-matchedcontacts. All of the lepromatous patientswere hypocalcemic, but plasma levels ofionizcd calcium and the acid-base statuswere normal. The average daily food intakeassessed through a questionnaire revealedadequate nutrition of patients and controls.Plasma proteins and 1,25-dihydroxyvita-min D, and intestinal absorption ofcalciumwere discarded as the causes of the hypo-calcemia.

In Nitro experiments designed to inves-tigate the effect of hydrogen ion concentra-tion on the equilibrium between calciumion and proteins revealed that, at normalpH values, plasma proteins from lepro-matous leprosy patients bind a smaller frac-tion of total plasma calcium than those fromcontrols. This phenomenon produces a nor-mal concentration of ionized calcium thatdetermines a normal parathyroid status asindicated by the normal urinary excretionof hydroxyproline and plasma concentra-tions of alkaline phosphatase (total and boneisoenzyme) and tartrate-resistant acid phos-phatase.

RESUMENEste trabajo reporta un estudio realizado en 10 pa-

cientes lepromatosos ambulatorios y en 10 de sus con-tactos apartados por edad y sexo. Todos los pacienteslepromatosos fueron hipocalcémicos pero los nivelesplasmáticos de calcio ionizado y el estado ácido-basefueron normales. El consumo diario de alimentos, es-tablecido a través de un cuestionario, fue adecuadotanto en los enfermos como en sus contactos. Las pro-teínas plasmáticas y la 1,25-dihidroxivitamina D3, asicomo la absorción intestinal de calcio, fueron descar-tadas como causas de la hipocalcemia.

Los experimentos in ritro disenados para investigarel erecto de la concentración del ión hidrógeno sobreel equilibrio entre el ión calcio y las proteínas, reve-laron que a valores normales de pH las proteínas plas-máticas de los pacientes lepromatosos enlazan una me-nor fracción del calcio total que las proteínas plasmáticasde los controles. Este fenómeno produce una concen-tración normal de calcio ionizado que determina unestado paratiroideo normal, según se deduce de la ex-creción urinaria normal de hidroxiprolina y de Ias con-centraciones en plasma de fosfatasa alcalina (la total y

590^ International .1ournal of Leprosy^ 1993

la isoenzima ósea) y de la fosfatasa ácida resistente altartrato.

RESUMECet article relate une étude réalisée parmi 10 patients

lépromateux externes et le mime nombre de contactsappariés pour l'áge et Ir. sexe. Tous les patients lépro-mateux étaient hypocalcémiques, mais les taux plas-matiques d'ions calcium et le statut acide-base étaientnormaux. L'absorption quotidienne moyenne de nour-riture évaluée à l'aide d'un questionnaire a montré unenutrition adéquate des patients et des témoins. On aécarté les protéines plasmatiques, la 1,25-dihydroxy-vitamine D, et l'absorption intestinale du calciumcomme causes de l'hypocalcémie.

Des expérimentations in nitro réalisées pour analyserl'effet de la concentration en ion hydrogène sur l'équi-libre entre l'ion calcium et les protéines ont montréqu'à des valeurs normales du pH, les protéines plas-matiques provenant de patients lépromateux se lient àune part plus petite du calcium plasmatique total quecelles provenant des témoins. Ce phénomène produitune concentration normale de calcium ionisé qui dé-termine un statut parathyroïdien normal comme l'in-diquent l'excrétion urinaire normale d'hydroxyprolineet les concentrations plasmatiques de phosphatase al-kaline (totale et l'isoenzyme osseux) et de phosphataseacide résistant au tartrate.

AcknoHiedgment. A grant from the Consejo Na-cional de Investigaciones Cientificas y Técnicas par-tially supportcd this work. Dra. Adriana S. Dusso(Washington University', St. Louis, Missouri U.S.A.)kindly measured the plasma leveis of 1,25-dihydroxy-vitamin D.

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