THE PARATHYROIDGLANDSAND PHOSPHORUSHOMEOSTASIS12

By JOHND. CRAWFORD,MAURICEM. OSBORNE,JR., NATHANB. TALBOT,MARYL. TERRY, AND MARYF. MORRILL

(From the Children's Medical Service, Massachusetts General Hospital, and the Department ofPediatrics, Harvard Medical School)

(Submitted for publication January 10, 1950; accepted, July 28, 1950)

INTRODUCTION

It has long been appreciated that certain of theendocrine glands are essential to the maintenanceof a healthy internal environment. For a timeit was' assumed that the endocrine systems main-tained health by producing their hormones at ap-proximately constant rates. However, as sensi-tive indices of endocrine activity have becomeavailable, it has become increasingly clear thathormone production rates are varied over a widerange in accordance with physiologic homeostaticneeds.

Considerable information concerning relationsbetween parathyroid activity and phosphorusmetabolism is available (1-12). The presentpaper attempts to extend such observations bydescribing relations between phosphorus intake,serum phosphorus concentration 3 and parathyroidactivity. The early sections of this paper areconcerned primarily with studies designed to in-dicate how variations in parathyroid activity maybe recognized. In latter sections, information con-cerning homeostatic alterations in parathyroid ac-tivity is presented.

The studies reported here are concerned almostexclusively with the influence of the parathyroidglands on phosphorus metabolism. While changesin calcium metabolism have also been considered inthe progress of the investigation, such changesappear to be separable from a consideration of thechanges in phosphorus metabolism.

MATERIAL AND METHODS

A. Animal experiments. Experiments were conductedon two groups of young, growing male rats of the Wistar

1 This work was supported in large measure by grantsfrom the Commonwealth Fund of New York.

2Read before the Society for Pediatric Research, May,1950.

8 The term, "serum phosphorus concentration," is usedhere and subsequently to refer to serum inorganic phos-phorus concentration.

strain ranging between 175 and 300 gm. in body weight.Individual animals were kept in separate small metaboliccages so that dietary intake and urine output could beaccurately quantitated. One group of animals was para-thyroidectomized by removal of the parathyroid glandsalone or in combination with the thyroid under nembutaland ether anesthesia; the other group served as intactcontrols. In the thyroparathyroidectomized animals, theeffects of thyroid removal were offset by the inclusion of10 mgm. of desiccated thyroid in the daily ration as recom-mended by Rand and Riggs (13).

All animals were fed daily a diet consisting of 16 gm.per animal of an essentially phosphorus free but other-wise complete synthetic diet4 fortified with known amountsof ammonium phosphate [(NH4) 2HPO.] solution.Measurements of plasma inorganic phosphorus concen-tration and urinary phosphorus excretion were made atsix different levels of phosphorus intake. A period of atleast six days was permitted on any given regime beforecollections of blood and urine samples were made. Bloodsamples, 0.8 cc. in volume, were obtained under oil withthe aid of an heparinized syringe by cardiac puncture af-ter light ether anesthesia. Urine samples were collectedover periods of 24 hours and were treated with trichlora-cetic acid to precipitate protein.

Glomerular filtration rate in both groups was estimatedfrom endogenous creatinine clearance measured in fourto eight animals on each of the various dietary regimes.Clearances were based on the creatinine content of 24-hour urine specimens and end-point plasma samples.The average values for creatinine clearance thus ob-tained were taken as indices of the glomerular filtrationrate of comparable animals on similar diets.

4Composition of synthetic low phosphorus diet (modi-fied from Deane, and associates 114]): CaCOs, 37.50 gm.;NaCl, 11.70 gm.; MgSO4 7 H20, 25.40 gm.; NaI, 0.0075gm.; CuSO,-5 H,O, 0.10 gin.; ZnCl2, 0.022 gm.; MnSO4-4 H,O, 0.81 gm.; FeC.HsO7-3 H20, 7.40 gm.; KCI, 7.15gm.; NaHCO,, 45.50 gm.; Cystine, 5.0 gm.; Choline, 2.5gm.; Thiamine, 0.010 gm.; Riboflavin, 0.030 gm.; Pyri-doxine, 0.010 gm.; Sodium Pantothenate, 0.062 gm.; To-copherol, 0.100 gm.; Menadione, 0.125 gm.; Nicotinicacid, 0.125 gm.; Inositol, 2.5 gm.; para-aminobenzoicacid, 5.0 gm.; Vitamin A, 65,000 I.U.; Vitamin D, 13,-000 I.U.; Washed Casein, (Devitaminized casein; Shef-field Farms, Inc.), 625.0 gm.; Corn Oil (Mazola, CornProducts Refining Co.), 125.0 gm.; Dextrose, 1,910.0 gm.Each 16 gm. of this diet was found by analysis to con-tain approximately 15 mgm. of phosphorus.

1448

THE PARATHYROIDGLANDSAND PHOSPHORUSHOMEOSTASIS

Operated animals in which there was histologic or

functional evidence of incomplete parathyroid, removal

were discarded from the series. Representative intactanimals of the control group were sacrificed at each dif-ferent level of phosphorus intake after completion of bloodand urine collections. The parathyroid glands of theseanimals were studied histologically to relate gland size,cell density and changes in cytology with dietary phos-phorus intake.

B. Human experiments. The subjects of all but one

of the human experiments were healthy adult males.Subject J. T. of Experiment II, Figure 4, was an essenti-ally normal 10-year-old girl recovering from a minorelective surgical procedure. Phosphorus was adminis-tered to this patient intravenously. The other subjectstook basal diets of known phosphorus content. Duringcertain experimental periods the phosphorus content ofthe diet was augmented by the addition of neutral phos-phate salt mixtures. In others the phosphorus availablefor assimilation was diminished by adding aluminumhydroxide gel to the diet. Details of the various dietsand intravenous fluids employed are given in connectionwith the individual experiments.

Urine collections for clearance studies were obtainedby spontaneous voidings under conditions of moderatediuresis (3-S5 cc. per minute). Periods ranged from20 to 120 minutes in length. Venous blood samples were

drawn at the beginning and end of each clearance periodunder 90 minutes. An additional blood sample was ob-

tained at about 60 minutes when periods exceeded 90minutes in length. Midpoint values were assumed toequal the mean of the values obtained on these samples.

C. Analytical methods and calculations. Analyses ofurine and serum or plasma for inorganic phosphorus con-

centration were carried out by the method of Fiske and

,Subbarow (15). Calcium analyses were done,according tothe method of Fiske and Logan (16); serum protein bythe method of Linderstrom-Lang as modified by Lowryand Hastings (17). Serum ionized calcium concentra-tion was estimated from the nomogram of McLean andHastings (18). Analyses for "apparent creatinine" were

performed by a modification.5 of the method of Bonsnesand Taussky (19).

Creatinine clearances were calculated by the formula,

5The creatinine method of Bonsnes and Taussky was

modified for the Evelyn colorimeter as follows: Theapparatus was set at the 6 cc. aperture using the "colorim-eter light brighter" control. A "blank" containing 4 cc.

of distilled water, 1 cc. of 1.0% sodium picrate and 1 cc.

of 0.75 N NaOHwas inserted and the galvanometer ad-justed to "100" on the scale. A density curve was con-

structed for known amounts of creatinine ranging from0.5 to 50 micrograms per 6 cc. sample. Analysis ofstandard solutions showed the most sensitive portion ofthe curve to lie in the range 0.5-12 micrograms per 6 cc.

sample. Accordingly, urine samples for analysis were di-luted to bring the creatinine content of 4 cc. of dilutedurine to between 1 and 12 micrograms.

For colorimetry, matched tubes were placed in a waterbath at 26.00 C. These were filled with 4 cc. of dilutedurine and 1 cc. of 1% sodium picrate. Exactly 10 min-utes before reading in the colorimeter, 1 cc. of 0.75 NNaOHwas added.

Serum analysis was conducted on the water clear su-

pernatant obtained after centrifuging 1 cc. of serum towhich had been added 8 cc. of N/12 H2SO4 and 1 cc. of10% sodium tungstate. To 4 ml. of supematant the so-

dium picrate and sodium hydroxide reagents were addedas for urine. Similarly, color development was allowedto proceed at 260 C. for 10 minutes before reading.

BLE I

Effect of changes in dietary phosphorus intake on intact and parathyroidectomized rats

Dietary regimen Plasma inorganic phos- Urinary phosphorus Creatinine clearancephorus concentration excretionNo. ingroup

Doet. Phosphios Average Std. dev. Average Std. dev. Average Std. dev.

mgm.f sq. m./ mgm.Isq. M.- mgm.Isq. m./ liters/sq. m./ liters/sq. m./24 hrs. mgm./100 cc. mgm./100 cc. 24 hrs. 24 hrs. 24 hrs. 24 hrs.

Intact 21 (4)* I 435 5.55t 0.63 4 1.20 35.8 2.2rats 20 (8) III 1615 6.08 1.04 338 77 44.5 5.1

14 (4) IV 2780 6.11 0.72 1110 425 43.7 5.012 (4) V 5130 6.29 1.13 1816 602 43.0 7.18 (4) VI 7770 7.08 1.25 3533 838 53.4 4.0

Parathyroid- 14 (4) I 478 6.15 1.28 4 1.64 41.2 3.2ectomized 12 (4) II 825 8.88 1.41 72 10.7 41.7 6.6

rats 14 (6) III 1815 11.81 1.40 305 133 32.9 1.6

* The figures in parentheses in this column refer to the number of animals in which measurements of creatinineclearance were made.

t The values for plasma phosphorus concentration given in the table are those obtained after approximately sevendays of constant phosphorus intake. Values were not observed to vary from these levels when the various diets weregiven for longer periods, except in the case of intact animals on diet I. On this diet, presumably because of its verylow phosphorus content, progressive hypophosphatemia developed. Plasma phosphorus concentrations observed in theintact animals after two and three weeks on diet I averaged 2.90 and 1.97 mgm. %, respectively.

1449

J. CRAWFORD,M. OSBORNE, JR., N. TALBOT, M. TERRY, AND M. MORRILL

Cl = (U V)/P (20). The values obtained for ratsare in good agreement with measurements of glomerularfiltration rate estimated on the basis of inulin, mannitoland exogenous creatinine clearance recorded in the litera-ture (21-24). Similarly, the values for endogenouscreatinine clearance found in normal adult human sub-jects are comparable with those reported by Brod andSirota (25) and Sirota, Baldwin and Villarreal (26) indemonstrations of the close parallelism between endoge-nous creatinine and inulin clearances in healthy adults.

Phosphorus appearing in glomerular filtrate (G.F.P.)was calculated as the product of glomerular filtration rate

and plasma or serum inorganic phosphorus concentra-tion (27, 28). Phosphorus reabsorbed by the renal tu-bules (T.R.P.) was found as the difference betweenglomerular filtrate and urinary phosphorus (U.P.) (27).In certain sections of the manuscript the ratio, T.R.P./G.F.P., is used to indicate the portion of phosphorus fil-tered at the glomerulus which is reabsorbed by the renaltubules.

Surface area of the rat was computed from the formulaof Benedict: Surface Area (sq. m.) = 0.00092 -.4 [Weightin grams]' (29). Surface area of human subjects wasestimated from the nomogram of Boothby and Sandiford

3 4 5 6DIETARY PHOSPHORUSINTAKE

GM/SQ.M/24 HRS.

FIG. 1. THE INFLUENCE OF CHANGESIN DIETARY PHOSPHORUSINTAKE ONINTACT AND PARATHYROIDECTOMIZEDANIMALS

Dietary phosphorus intake is shown on the abscissa expressed as gm./sq. m./day.The ordinate of Section A gives plasma inorganic phosphorus concentration inmgm./100 cc. The shaded horizontal area encompasses the range of plasmaphosphorus concentration (2.5 x Std. Dev.) observed in normal animals ofcomparable age and strain fed complete synthetic diet, which provides a phos-phorus intake of approximately 3 gm. of phosphorus/sq. m./day. Observationsof plasma phosphorus concentration of normaf and parathyroidectomized ratsare given by the round and triangular points, respectively. The ordinate ofSections B and C give values in terms of mgm. of phosphorus/sq. m./day cor-responding to the heights of the vertical columns. The total height of eachcolumn indicates the value for glomerular filtrate phosphorus (G.F.P.); theshaded portion, phosphorus excreted in urine, (U.P.), while the open portion ofthe column indicates the value for phosphorus reabsorbed by the renal tubules(T.R.P.). The columns of Section B are constructed from observations on in-tact rats; those of Section C from observations on parathyroidectomized rats.

1450

THE PARATHYROIDGLANDSAND PHOSPHORUSHOMEOSTASIS

(30). For purposes of comparison all data are expressedper 1 sq. m. of body surface area.

RESULTS

A. The influence of changes in dietary phosphorusintake on intact and parathyroidectomizedanimals

Table I gives values for plasma inorganic phos-phorus concentration, urinary phosphorus excre-

tion and creatinine clearance of intact and parathy-roidectomized rats fed diets of varying phosphoruscontent. These data are presented graphicallyin Figure 1 in company with certain deriva-tives. Note in Section A that the intact animals

1.00 r-

0.80

0.60

T. RP.,/At;.F.P.

0

0

.0

0.40 F-

0

maintained plasma phosphorus concentrationswithin or close to the normal range on diets vary-ing in phosphorus content from 0.44 to 7.8 gm./sq. m./day. By contrast, the parathyroidectomizedanimals maintained normal plasma phosphorusconcentrations only on the diet providing less than0.5 gm./sq. m./day of phosphorus. They de-veloped marked hyperphosphatemia when dietaryphosphorus intake was increased to 0.8 gm./sq. m./day. Diets containing phosphorus in excess of1.8 gm./sq. m./day, well tolerated by normal ani-mals, resulted in food refusal, weight loss, tetanyand death in parathyroidectomized rats.

Sections B and C of Figure 1 set forth data de-rived as indicated in the section on methods. In

0

0

0

: ^XA .A£

e ALA~~~~~~~

00

INTACT ANIMALS

* Creatinine Clearance

No Creotinine Clearonce

PARATHYROIDECTOMIZEDANIMALS

A Creatinine Clearance

& No Creatinine Clearance0.20 I-

0

Og0

2.0 4.0 6.0 8.0 10.0 12.0 94.0

PLASMA INORGANIC PHOSPHORUSCONCENTRATION

mgm/lOOcc.

FIG. 2. RELATIONS BETWEENPLASMA INORGANIC PHOSPHORUSCONCENTRATIONAND THE RATIo, T.R.P./G.F.P.The abbreviations used correspond to those given in the legend of Figure 1. Values for plasma inorganic

phosphorus concentrations are given on the abscissa and for the ratio, T.R.P./G.F.P., on the ordinate. Thepoints enclosed in circles are calculated from observations on intact rats; those enclosed in triangles from obser-vations on parathyroidectomized rats. The dark points represent observations on animals in which measure-ments of endogenous creatinine clearance were obtained simultaneously with the measurements of phosphorusin plasma and urine. The open points represent observations on animals in which the average creatinine clear-ance of similarly prepared animals on the same diet was used as an index of glomerular filtration rate. TheRoman numerals adjacent to the crosses connected by straight lines refer to the average values found for ani-mals receiving the dietary regimes of corresponding number as described in Table I.

145-1

I I 1 I I

J. CRAWFORD,M. OSBORNE, JR., N. TALBOT, M. TERRY, AND M. MORRILL

intact animals (Section B) it is seen that glomeru-lar filtrate phosphorus increased sharply from a

value of approximately 2.0 gm./sq. m./day whenrats were fed the diet providing the least phos-phorus to a nearly constant value of about 2.7 gm./sq. m./day in rats fed the three diets providingphosphorus in moderate to large quantities. Afurther, sharp rise in glomerular filtrate phos-phorus to approximately 3.8 gm./sq. m./day oc-

curred in animals receiving the diet containing thelargest quantity of phosphorus. In parathyroid-ectomized animals (Section C) glomerular filtratephosphorus had a value of approximately 2.5 gm./sq. m./day when animals were fed the lowestphosphorus diet. This value increased abruptlyto 3.7 gm./sq. m./day when animals were given a

diet intermediate in phosphorus content; a fur-ther small increase to approximately 3.9 gm./sq.m./day was observed in parathyroidectomized

rats fed the diet supplying the largest amount ofphosphorus tolerated. When intact and para-

thyroidectomized animals were fed diets of com-

parable phosphorus content, much higher valuesfor glomerular filtrate phosphorus were observedin the operated than in the intact group.

The urinary phosphorus excretion of intact ani-mals (Section B) increased from very small quan-

tities when the rats were fed the diet providing theleast phosphorus to 3.5 gm./ sq. m./day when thediet providing the greatest amount of phosphoruswas given. On comparable intakes of phosphorus,parathyroidectomized animals (Section C) ex-

creted approximately the same quantities of phos-phorus in the urine as intact animals.

Tubular reabsorption of phosphorus was cal-culated to have a value of approximately 2.0 gm./sq. m./day when intact animals (Section B) re-

ceived the diet providing the least phosphorus.

TABLE II

The effects of parathyroid extract* (P.E.) administration

Perio | Time Midpoint Ur|inay P Creatinine iPerod Time Midpoint |Urnay P| Creatinineelapsed serum P clearance elapsed serum

P laac

mgm./sq. m./ cc./sq. m./ mgm./sq. m./ cc./sq. M./Xio.j his. mgm./100 cc. Z4 his. 24 hrs. no. hrs. mgm./100 cc. 224 hrs. 24 hrs.

Experiment I. Subject J. D. C., Normal diet. Over- Experiment II. Subject M. O., Normal diet. Over-night fast extended throughout experimental period. night fast extended throughout experimental period.100 units P.E. by vein immediately after period No. 4. 200 units P.E. by vein immediately after period No. 4.

1 0.5 3.10 344 1 0.5 3.20 4062 1.0 3.15 500 2 1.0 3.46 3713 1.5 3.22 516 96,300 3 1.5 3.59 482 113,0004 2.0 3.33 536 4 2.0 3.59 5925 2.5 3.43 870 5 2.5 3.64 980 1.6 3.0 3.27 771 1| 0,000 6 3.0 3.90 955 115,5007 3.5 3.27 563 1000 7 3.5 4.03 7438 4.0 3.30 576 8 4.0 4.05 725 } 1,09 4.5 3.15 511 99 |08 9 4.5 4.28 725 112000

10 5.0 3.23 485 ,000 10 5.0 4.28 768 114,50011 5.5 3.43 579 97,300

Experiment III. Subject W. L., Low phosphorus Experiment IV. Subject W. L., High phosphorusdiet.t Overnight fast extended throughout experi- diet. Diet of experiment III supplemented for fourmental period. 200 units P.E. by vein immediately days by neutral mixture of potassium phosphate saltsafter period No. 2. to give 1.7 gm./sq. m./day of phosphorus. Over-

night fast extended through experimental period. 200units of P. E. by vein immediately after period No. 2.

1 1 2.62 18 99,300 1 1 2.87 500 93,6002 2 2.67 54 95,200 2 2 3.05 431 96,0003 3 3.15 304 102,750 3 3 3.17 735 107,3004 4 3.51 457 101,400 4 4 3.23 736 93,400S 5 3.72 435 92,700 S 5 3.49 952 89,600

* Weare indebted to Eli Lilly and Co. for furnishing the parathyroid extract Lot No. 4077-440185 used in theseexperiments.

t This diet was constructed to give approximately 0.2 gm./sq. m./day of phosphorus. It had been taken for four daysprior to this experiment.

1452

THE PARATHYROIDGLANDSAND PHOSPHORUSHOMEOSTASIS

EXPERIMENT1. EXPERIMENTR. EXPERIMENTm.Subpct JD.C.l Subject KO. Subject WL.

LOWPHOSPHORUSUSUtAL DIRT USUALDIET DIET

Sectia A

TRPMG/SQM./24HRS.

SWc""n *.u

0.95

0.90

TRP/GFP0.85

Q06

0.78

.70

0.6

060

0.58

EXPERIMENTN1.Subject WL.

HIGH PHOSPHORUSDIET

Parothyroid Extract Parathyroid Extract Parathyroid Extract Parathyroid Extract

4000 40/sq00lo /sq 0/MSsq@ 400

3500 3&500

3000 i - 3000

2500 2500

2000 .LLJ.L1L JjJJJJ .L4~~4I~4, 2000

095

0.90

0.85

0.80

0.5

0.70

065

0.60

055

-2 -1 0 1 2 3-2 -1 0 1 2 3-2 -1 0 1 2 3 -2-10 1 2

HOURS

FIG. 3. THE EFFECT OF PARATHYROIDEXTRACTON T.R.P. AND THERATIO,T.R.P./G.F.P.

The abbreviations used correspond to those given in the legend of Figure 1. InSection A are plotted values for T.R.P. expressed as mgm. of phosphorus/sq. m./24 hrs.Open circles represent values oblained before, closed circles values obtained after in-travenous parathyroid extract administration. Time in hours is given on the abscissae.

Section B gives values for the ratio, T.R.P./G.F.P. Open circles again representpreinjection values, closed circles postinjection values. The abscissae are as forSection A.

This value rose to approximately 2.4 gm./sq. m./day with the first increment in dietary phosphorus,but then underwent a progressive fall as dietaryphosphorus was further increased. The value de-clined to a figure of but 0.2 gm./sq. m./day in in-tact animals fed the diet of highest phosphoruscontent. In the parathyroidectomized group (Sec-tion C) the value for tubular phosphorus reabsorp-tion was 2.5 gm./sq. m./day when the diet pro-

viding the least phosphorus was given and rose toapproximately 3.6 gm./sq. m./day in animals fedthe two diets containing phosphorus in largeramounts. On similar diets, the values for tubularreabsorption of phosphorus were distinctly higherin operated than in intact animals.

Figure 2 sets forth the data of Table I and

Figure 1 in another manner. Here values forthe ratio, T.R.P./G.F.P. (ordinate), are plottedagainst values for plasma phosphorus concentra-tion (abscissa). It is seen that the ratio valuesobtained for intact animals fell from approximately1.0 to 0.0 as the phosphorus intake was increasedfrom the lowest to the highest levels. By con-

trast, the ratio values found for the parathyroid-ectomized animals varied but little and rangedbetween 0.99 and 0.88. While the fall in ratiovalue observed in intact rats occurred over a rela-tively narrow range of plasma phosphorus concen-

tration, the high values observed in parathyroidec-tomized animals were maintained over a consider-able range of plasma phosphorus concentration.

These data show that when phosphorus intake

2 -I o I 2 3 -2 -1 o I 2 32 1 o I z 3 'z v ,

Jr ii

n r

J u

1453

I

J. CRAWFORD,M. OSBORNE, JR., N. TALBOT, M. TERRY, AND M. MORRILL

is varied, the intact animal has the capacity tomaintain plasma phosphorus concentration nearlyconstant by suitable adjustments in renal phos-phorus clearance. This capacity is lost after para-thyroidectomy. The data also show that absolutevalues for renal tubular phosphorus reabsorptionare widely variable both in intact and in parathy-roidectomized animals. On the other hand, thoughthe relationship between phosphorus filtered at theglomerulus and phosphorus reabsorbed by the tu-bules (T.R.P./G.F.P.) also varies in intact ani-mals, it remains essentially constant after the para-thyroid glands have been removed.

These observations suggest that the value forT.R.P./G.F.P. is determined to a major extent by

parathyroid hormone. Stated differently, theysuggest that parathyroid hormone determines theproportion of glomerular filtrate phosphorus re-absorbed by the renal tubules. To gain furtherinformation on this point, studies on the effectsof administering parathyroid extract to human be-ings were carried out.

B. The effect of parathyroid extract on normnal hu-man beings

Table II gives data obtained on three normaladults to whom parathyroid extract was adminis-tered. In Figure 3, the derived values for phos-phorus reabsorbed by the kidney tubules (T.R.P.)(ordinate, Section A) and for the ratio, T.R.P./

FIG. 4. THE IMMEDIATE EFFECTS OF ABRUPT INCREASES IN PHOSPHORUSINTAKE

The abbreviations used are the same as those given in the legend of Figure 1. In Section A of both experi-ments, the ordinate gives values for serum phosphorus concentration. The ordinate of Section B gives valuesfor G.F.P., T.R.P. and U.P. represented by the sectors of the columns. The value for the broken line represent-ing phosphorus intake is also shown by this ordinate. In Section C the ordinate gives values for the ratio,T.R.P./G.F.P. The abscissa in both experiments shows time in hours.

1454

Vp

I --I I I

THE PARATHYROIDGLANDSAND PHOSPHORUSHOMEOSTASIS

G.F.P. (ordinate, Section B), are presentedgraphically. It is seen in Section A that adminis-tration of parathyroid extract failed to promptany consistent change in the absolute values forT.R.P. The values were irregular in ExperimentI; an initial fall followed by a sharp rise was seen

in Experiment II; in Experiment III the post-injection values rose while in Experiment IV thevalues declined. On the other hand, it is evidentin Section B that parathyroid extract administrationwas promptly and consistently followed by a very

significant decrease in the value for T.R.P./G.F.P. This occurred irrespective of the positionof the control points (compare Experiments IIIand IV). In Experiments I and II the values forthe ratio returned to control levels within approxi-mately three hours after parathyroid extract ad-ministration. A similar return to control valuespresumably would have occurred had the observa-tions in Experiments III and IV been continuedover longer periods.

These experiments like those of Figure 1 fail toshow any consistent relation between absolute val-ues for T.R.P. and parathyroid hormone status.

On the other hand, they do show a consistenttendency for the value for T.R.P./G.F.P. to de-cline under conditions of increased parathyroidhormone concentration.

The foregoing observations suggest stronglythat parathyroid hormone is the major determinantof the ratio, T.R.P./G.F.P. When parathyroidhormone is absent (parathyroidectomized rats,Figure 2), the ratio becomes fixed at a value ap-

proaching unity. When parathyroid extract isadministered, the value of the ratio falls towardszero. It follows that the value for T.R.P./G.F.P.should serve as an approximate index of para-thyroid hormone activity. Experiments designedto test this thesis are presented in the subsequentsections.

C. The effect of changes in phosphorus intake on

serum phosphorus concentration, urinaryphosphorus excretion and on the ratio,T.R.P./G.F.P., in normal human subjects

1. The immediate effects of abrupt increases inphosphorus intake. Data obtained from two sub-jects whose intakes of phosphorus were suddenly

TABLE III

The immediate effects of abrupt increases in phosphorus intake

Period Telas serumMp Urinary P Creatinine Period Time Midpoint Urinpy | Creatielapsedserum P clearance elapsed serum P UrnyP clearance

mgm./sq. m./ cc./sq. m./ mgm./sq. m./ cc./sq. m./no. min. mgm./100 cc. 24 hrs. 24 hrs. no. min. mgm./100 cc. 24 hrs. 24 hrs.

Experiment I. Subject M. M. 0. Low phosphorus Experiment II. Subject J. T. Ordinary diet discon-diet prior to experiment. Neutral sodium phosphate tinued 12 hours prior to first experimental period.solution* taken by mouth at 0.5 hour intervals begin- I.V. glucose and water started six hours before experi-ning at end of period No. 3. ment and continued throughout. Neutral phosphate

solutiont added to infusion fluid at end of periodNo. 3.

1 15-60 3.75 335 125,000 1 60 2.97 2002 106 3.66 193 109,000 2 210 2.89 165 67,2403 205 3.45 169 113,000 3 270 2.75 1744 282 4.71 985 103,000 4 430 3.26 3925 344 5.06 1013 99,000 5 510 3.77 1267 77,3606 450 5.12 1830 103,000 6 630 3.99 800 l7 540 5.58 2042 106,000 7 750 4.90 1844 66,3008 630 6.02 2425 95,000 8 870 5.37 27109 690-742 5.97 2621 101,000 9 990 5.25 2770 85,800

10 790 5.60 2688 107,000 10 1275 4.80 2480 60,50011 846 5.35 2675 117,000 11 1470 4.34 2120 64,22012 910 5.12 2305 119,000

* Composition of phosphate solution ingested: 1.0 gm. Na2HPO4and 0.53 gm. NaHPO4/L. of which subject' took230 cc. every 0.5 hr. By analysis the phosphate content of this solution was found to be 35.4 mgm./100 cc.

t Infusion fluid control periods: Na, 29 mEq/L.; K, 17 mEq/L.; Cl, 22 mEq/L.; Lactate, 23 mEq/L.; Dextrose,10 gm./100 cc.; pH, 7.45. Infusion fluid phosphate periods: Na, 27 mEq/L.; K, 17 mEq/L.; P, 64.8 mgm./100 cc.;Lactate, 8 mEq/L.; Dextrose, 10 gm./100 cc.; pH, 7.42. The Na, K, Cl, P and pH values of these solutions were deter-mined by chemical analysis.

1455

J. CRAWFORD,M. OSBORNE, JR., N. TALBOT, M. TERRY, AND M. MORRILL

TABLE IV

The ultimate effects of changes in phosphorus intake*

Dietary Serum Urine Creatinine Serum Serum Serum

phosphorue phosphorus phosphorus clearance calcium protein oalciumgm./s.m./ mem./sq. m./ cc./sq. m./

24 hrs. mgm./100 cc. 24 hrs. 24 hrs. mgm./100 cc. gm./100 cc. mgm./100 cc.Experiment I 0.2 3.45 84 93,250 _Subject H. Kt 0.8 3.99 374 112,100

2.4 4.68 1,697 115,725 - -

Experiment II 0.1 2.85 33 112,000 8.2 7.2 3.5Subject M. 0t 0.3 3.87 143 110,250 8.7 7.6 3.6

3.3 4.75 2,620 110,000 8.3 6.8 3.7

Experiment III 0.2 2.65 36 97,250 9.8 7.2 4.2Subject W. L.I 1.7 3.23 1,067 88,470 10.3 7.1 4.5

* The figures for serum phosphorus and calcium concentration, urine phosphorus excretion and creatinine clearanceare averages calculated from multiple determinations. All subjects received their different dietary phosphorus intakesfor a minimum of four days before these measurements were made.

t With the aid of a dietitian a basic diet was constructed which contained approximately 0.8 gm./sq. m./day ofphosphorus. This diet was taken throughout the experiment. Phosphorus assimilation was reduced to approximately0.2 gm./day by the taking of 45 cc./sq. m./day of aluminum hydroxide gel (Amphogel). An increase in the phosphoruscontent of the diet was brought about by adding 1.6 gm./sq. m./day of phosphorus in the form of 1.37 gm. of KH2POGand 5.73 gm. of Na2HPO4.

$ For this subject a basic diet was constructed to contain approximately 0.3 gm./sq. m./day of phosphorus whichwas taken throughout the experiment. Assimilation of phosphorus was reduced to approximately 0.1 gm./sq. m./dayby the taking of 50 cc./sq. m./day of aluminum hydroxide gel. The phosphorus content of the diet was increased toapproximately 3.3 gm./sq. m./day by addition of 2.63 gm. of KH2PO4and 11.0 gm. of Na2HPO.

§ For subject W. L. the basic diet was constructed to contain approximately 0.2 gm./sq. m./day of phosphorus whichwas taken throughout the experiment. The phosphorus content was increased by supplementing the basic diet with1.5 gm./sq. m./day of phosphorus by addition of 1.32 gm. of K2HPOand 6.75 gm. of KH2PO4.

increased by the oral or intravenous administra-tion of a neutral mixture of phosphate salts aregiven in Table III.

These data and the derivative values for phos-phorus filtered at the glomerulus, phosphorus re-absorbed by the tubules and for the ratio, T.R.P./G.F.P., are set forth graphically in Figure 4.Section A of this figure shows that in both ex-periments values for serum phosphorus concen-tration commenced to rise immediately after thebeginning of phosphate administration. This risecontinued during the early periods of observation,a peak being reached after about 12 hours of con-tinuous phosphate loading. Thereafter, a fall inserum phosphorus concentration toward the pre-administration control level was seen. Section Bshows the relations between glomerular filtratephosphorus, urine phosphorus and phosphorus re-absorbed by the kidney tubules. In both experi-ments glomerular filtrate phosphorus was observedto increase. In Experiment II, in which observa-tions were made over a longer period of time, thisincrease was followed by a secondary fall. Urinephosphorus increased rapidly during the earlyhours of both experiments, then continued at high

levels and finally declined slightly. The valuesfor phosphorus reabsorbed by the tubules showedlittle change in Experiment I, while in Experi-ment II the values decreased quite markedly asthe phosphorus load was maintained. Section Csets forth values for the ratio, T.R.P./G.F.P. Thefirst change noted in both experiments was arapid decline from relatively high control values.In the final hours of phosphate administration, asecondary tendency for T.R.P./G.F.P. to stabilizeat new low values is suggested.

In the next section these observations are ex-tended to show ultimate adaptation to changes inphosphorus intake.

2. The ultimate effects of changes in phosphorusintake. For control purposes, three normaladults were placed on diets of essentially constantphosphorus content for periods of at least fourdays. On the final day of this regimen, serumphosphorus concentration, urinary phosphorus ex-cretion and endogenous creatinine clearance weremeasured at frequent consecutive intervals overperiods of eight to 16 hours. Diets providing acontrasting level of phosphorus intake were thengiven for a second period of at least four days. At

1456

THE PARATHYROIDGLANDSAND PHOSPHORUSHOMEOSTASIS

the end of each such period the aforementionedmeasurements were repeated. The results ob-tained are presented in Table IV and Figure 5.

Section A of Figure 5 describes relations be-tween serum phosphorus concentration (ordinate)and dietary phosphorus intake (abscissa). Al-though increases in phosphorus intake regularlyresulted in increases in serum phosphorus concen-tration, the serum phosphorus concentration wasevidently not a direct function of dietary phos-phorus intake. This is particularly well shown inExperiment II. Here the relatively small firstincrease in dietary phosphorus intake resulted ina rise of 1 mgm./100 cc. in the concentration ofserum inorganic phosphorus. Following a second,tenfold increase in phosphorus intake, the serumphosphorus concentration rose only 0.9 mgm./100Cc.

In Section B of this figure the relation of dietaryintake to urinary excretion, renal tubular reabsorp-tion and glomerular filtration of phosphorus areshown in the familiar columnar diagrams. It isseen that the values for glomerular filtrate phos-phorus bear essentially the same relations to dietaryphosphorus intake as do those for serum phos-phorus concentration described above. Urinaryphosphorus output was very nearly directly pro-portional to dietary intake, approximately 75%oof the ingested phosphorus being excreted bythe kidneys. Values for tubular phosphorus re-absorption were quite variable. In ExperimentII, when dietary phosphorus intake was increasedfrom 0.1 to 0.3. gm./sq. m./day, a 25%o increasein tubular reabsorption of phosphorus occurred.When phosphorus intake was further increased to3 gm./sq. m./day, the value for phosphorus re-

Secon A 5.0

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FIG. S. THE ULTIMATE EFFECTS OF CHANGESIN PHOSPHORUSINTAKEThe abbreviations used correspond to those given in the legend of Figure 1. The ordinate of Section A gives

serum phosphorus concentration. In Section B the ordinate gives values for G.F.P., T.R.P. and U.P. as repre-sented by the sectors of the columns. In Section C, the value for the ratio, T.R.P./G.F.P., is given. The abscis-sae show dietary phosphorus intake in gm./sq. m./day.

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1457

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J. CRAWFORD,M. OSBORNE, JR., N. TALBOT, M. TERRY, AND M. MORRILL

absorbed by the tubules dropped 36 % to levelslower than those observed during prior dietaryregimes.

In Section C of the figure values for the ratio,T.R.P./G.F.P., are shown on the ordinate as theyrelated to dietary phosphorus intake. A diminu-tion in the value of the ratio was regularly ob-served with increments in dietary phosphorus;conversely, when dietary phosphorus intake wasrestricted, the value for the ratio consistently rose.

These changes in T.R.P./G.F.P. ratio shown inFigures 4 and 5 suggest that an increase in phos-phorus intake leads to a physiologic increase andthat a diminution in phosphorus intake leads tophysiologic decrease in parathyroid activity. Thisthesis finds support in direct observations on theparathyroid glands of the rats of Table I andFigure 1. The parathyroid glands of the animalsreceiving the highest phosphorus diet were ap-proximately four times larger than those of ratson the lowest phosphorus regime. In addition theparathyroids of rats given diets of high phosphoruscontent showed the chief cells to be closely packedwith an apparent reduction in interstitial tissue.Both the nuclei and cytoplasm were increased inamount and showed a tendency to stain morelightly than was the case in glands from animalsfed low phosphorus diets. In vivo, there was anapparent increase in vascularity of the hyperplasticglands. Similar relations between parathyroidsize and appearance and dietary phosphorus in-take have been noted by others (6-10, 12, 31, 32).

DISCUSSION

An extensive literature has been built up con-cerning the effects of administered parathyroidextract (4, 5, 12) and the effects of parathyroidgland extirpation (1, 3, 12, 32). These data indi-cate that a regular effect of administered extractis to increase renal phosphorus clearance (5, 12,33). In the absence of the parathyroid glands,phosphorus clearance declines to very low values(3, 12).

Several theories have been advanced to ex-plain the mechanism of these changes. For ex-ample, it has been suggested that parathyroidhormone may alter certain physical characteristicsof the phosphates in body fluids so as to render agreater fraction of the total blood phosphorus fil-terable at the glomerulus (12). Certain published

data, however, fail to support this thesis (27, 34,35) and in our own studies we have been unable todetect any significant change in the various moie-ties of total blood phosphorus either in sivo or invitro as a consequence of parathyroid extract ad-ministration. Harrison and Harrison (27) andPitts and associates (36, 37) have suggested thata renal tubular maximal capacity for phosphorusreabsorption (TmP) exists. This maximal ca-pacity or "threshold" value is presumed to be di-rectly under the influence of the parathyroid glands.These workers hold that the increased phosphorusclearance observed after parathyroid extract ad-ministration is a consequence of diminution in therenal tubular "threshold." Alternatively, Smith,Ollayos and Winkler (28) have proposed thatthe amount of phosphorus reabsorbed by the renaltubules is directly dependent at any given timeupon the amount of phosphorus being presentedthe tubules by glomerular filtration. In otherwords, they suggest that parathyroid hormonedetermines what proportion of the glomerular fil-trate phosphorus shall be reabsorbed by the renaltubules. This proportion is indicated by the ratio,T.R.P./G.F.P.

One would expect values representing the vari-able which is directly influenced by parathyroidhormone to become essentially fixed or constantwhen parathyroid hormone is lacking (parathy-roidectomy). Moreover, when parathyroid hor-mone concentration is increased as by parathyroidextract therapy, the values representing this vari-able should move consistently in one direction.Contrariwise, the values for the variable shouldalways move in the opposite direction when para-thyroid hormone is decreased (parathyroidec-tomy).

The experimental data presented above indicatethat values for T.R.P. remain changeable follow-ing parathyroidectomy (Figure 1) and undergoirregular changes when parathyroid extract isadministered (Figure 3). When G.F.P. valuesare varied by altering the phosphorus intake, val-ues for absolute T.R.P. also tend to vary (Figures4, 5).6 Thus T.R.P. measurements fail to fulfillthe requirements set forth above. Moreover, they

6 Such marked variations in absolute tubular reabsorp-tion values secondary to alterations in glomerular filtratevalues are not observed for such truly "threshold" sub-stances as glucose (38, 39).

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THE PARATHYROIDGLANDSAND PHOSPHORUSHOMEOSTASIS

do not suggest that control of renal phosphorus ex-cretion is accomplished by a tubular "threshold"mechanism.

On the other hand, it is seen that in parathyroid-ectomized animals the value for T.R.P./G.F.P. isessentially constant (Figure 2). Parathyroidextract administration consistently prompts a de-crease in T.R.P./G.F.P. (Figure 3). Finally,when phosphorus intake is appreciable, the valuesobtained in parathyroidectomized animals for theT.R.P./G.F.P. ratio are significantly higher thanvalues obtained on intact animals or human sub-jects taking comparable phosphate intakes (Fig-ures 2,4). Thus it appears that values for T.R.P./G.F.P. satisfactorily fulfill the criteria outlinedabove for a variable which is directly influencedby parathyroid hormone. It follows that theT.R.P./G.F.P. value should be useful as an ap-proximate, inverse, index of parathyroid activity.7

In the absence of parathyroid hormone the valuefor T.R.P./G.F.P. approaches unity (Figure 2).As parathyroid hormone concentration is increased,the value for T.R.P./G.F.P. falls towards zero(Figure 3). A zero value for this ratio representsmaximum parathyroid hormone action on the re-nal tubules (Figure 2). Recalculation of exten-sive clinical data from the literature has shown thatthe behavior of this index, T.R.P./G.F.P., is con-sistent with the presumed parathyroid status ofthe patients described.

Use of the T.R.P./G.F.P. index of the para-thyroid status of individuals under a variety of cir-cumstances shows the parathyroid glands of nor-mal individuals to be dynamic organs of homeo-stasis with respect to phosphorus metabolism.Adaptation to changing demands begins withina very short time after such changes are effectedand may be completed within as little as 12 hoursin certain instances (Figure 4). It defines in thenormal individual states of physiologic hypopara-thyroidism when phosphorus intake is low andstates of physiologic hyperparathyroidism whenphosphorus intake is high (Figures 5, 6).

7 While it is probable that factors such as pH andvitamin D influence the relation between T.R.P. andG.F.P. to some extent, presently available informationindicates that such factors are ordinarily of minor im-portance. It is also possible that the T.R.P./G.F.P.-parathyroid hormone relation breaks down when G.F.P.rises to higher levels than are encountered under ordi-nary clinical circumstances.

Though changes in dietary phosphorus intake mayprompt transient hyperphosphatemia as indicatedin Figure 4, they have not been found to cause anysignificant changes in serum ionized calcium con-centration values when adaptation to the new levelof intake has been completed (Table IV). Ac-cordingly, our data like that of Carnes and col-leagues (40), suggest that hyperphosphatemia aswell as hypocalcemia (41-43) constitutes a stim-ulus to parathyroid activity.

The present experiments shed light not only onthe homeostatic adjustments brought about by theparathyroid glands but also define certain limits ofadjustment. Whenphosphorus intake is markedlyrestricted urinary excretion of phosphorus dimin-ishes to essentially negligible quantities and theT.R.P./G.F.P. ratio attains maximal values.Under these circumstances, however, losses ofphosphorus from extracellular fluid to protoplasmand the skeleton may continue. When such meta-bolic demands for phosphorus are in excess ofthe quantities available from intake, the lowerlimit of adjustment with respect to serum phos-phorus homeostasis is exceeded. Hypophospha-temia is an obligatory consequence. An exampleof hypophosphatemia due to restriction in dietaryintake such that maximal renal phosphorus econ-omy fails to effect compensation is afforded by theintact animals fed low phosphorus synthetic diet.(See footnote 2, Table I.)

Contrariwise, when the phosphorus load is solarge that it cannot be eliminated quantitatively byreduction of the value for T.R.P./G.F.P. to zero,there is an obligatory rise in serum phosphorusconcentration. By prompting a corresponding in-crease in G.F.P. (and hence in urine phosphorus)this rise in serum phosphorus enables the organismto lessen tendencies to excess phosphorus reten-tion. In other words, urinary phosphorus excre-tion can be increased under conditions of constantT.R.P./G.F.P. (parathyroid activity) by elevatingserum phosphorus concentration.8 This mecha-

8 In our data (Tables I-IV) there is no evidence thatthe alternate possible mechanism for increasing urinaryphosphorus excretion, i.e., elevation of glomerular filtra-tion rate (44), is ordinarily a factor of importance. Aslight increase in creatinine clearance was regularly ob-served after parathyroid extract administration (TableII). It is to be noted, however, that the variations inurinary phosphorus excretion observed in these experi-ments are not a direct function of the variations in creati-

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J. CRAWFORD,M. OSBORNE, JR., N. TALBOT, M. TERRY, AND M. MORRILL

nism for increasing phosphorus elimination is em-ployed not only when the capacity of the para-thyroid hormone-T.R.P./G.F.P. mechanism hasbeen exceeded (parathyroidectomized animals ondiets II and III, Figure 1), but also during in-tervals when the parathyroids are becomingadapted to a change in phosphorus load (see earlyhours of Experiments I and II of Figure 4).

SUMMARYANDCONCLUSIONS

1. A relation has been demonstrated betweenthe ratio of phosphorus reabsorbed by the renaltubules to phosphorus in glomerular filtrate(T.R.P./G.F.P.) and parathyroid gland activity.The value for the ratio is:

A. Fixed close to unity by parathyroidectomy.B. Diminished by administration of parathyroid

extract.C. Inversely correlated with parathyroid gland

size.

These findings suggest that the value for theT.R.P./G.F.P. ratio may be used as an index ofparathyroid activity.

2. In normal individuals an increase in phos-phorus intake is followed within a few hours by afall in value of the ratio, T.R.P./G.F.P. Con-versely, shortly after phosphorus restriction, thevalue for the ratio begins to rise. These findingssuggest that the relative constancy of serum phos-phorus concentration in normal individuals ismediated to a major extent by homeostatic ad-justments in parathyroid activity.

3. Hyperphosphatemia per se appears to stimu-late increased parathyroid activity.

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THE PARATHYROIDGLANDSAND PHOSPHORUSHOMEOSTASIS

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