THE INDIRECT ASSESSMENT OF PARATHYROID FUNCTION

B. E. C. NORDIN and RUSSELL FRASER Postgraduate Medical School of London

THE phosphate-diuretic action of parathormone is not easy to measure, for even when phosphate excretion is expressed as a clearance this clearance still varies with the serum phosphorus level. The object of this paper is to show that changes in the activity of the parathyroid glands are associated with changes in phosphate clearance and how these are related to the serum phosphate concentration.

Milne, Stanbury and Thompson (1952) have already shown that when the serum phosphate is raised by the infusion of inorganic phosphate solutions there is, in normal subjects, a rise in the ratio of the phosphate clearance to the creatinine clearance (Cp/Ccr). They have assembled their data and those of Lambert, van Kessel and Leplat (1947) into a composite chart (Fig. 1) which shows the normal relationship between the serum and urinary phosphate. We have selected all the points on this composite chart up to a serum phosphate level of 8 mg. per 100 ml. and have taken the liberty of assuming that the regression in this part of the chart does not depart significantly from linearity. On this assumption, we have cal- culated the regression equation of these 117 points and found that it can be expressed as follows :

Serum P 20

- 0.05 . c,/ccr =

In other words, for each rise of 1 nig. per cent in the serum phosphate level the clearance ratio rises 0 . 0 5 , and the regres- sion line intercepts the C,/C,, axis at - 0 . 0 5 . The standard

222

Bone Structure and Metabolism G. E. W. Wolstenholme.Cecil,ia M. O'Con,ner

Copyright 0 1956 Ciba Foundation Symposium

INDIRECT ASSESSMENT OF PARATHYROID FUNCTION 223

deviation of the clearance ratio along this line is 0.06. Fig. 2 shows the relevant part of Milne, Stanbury and Thompson's chart, with the mean regression line and the 95 per cent limits drawn in by us.

W

8": 8 . . * . * . * I . X I

* * .. .. .. . x

X ... * * . . * .

**.. . . %

%. + * .'.*

0 . . *- .. 0 .

0 8 *;o ,:o... * * * 0 3 .

k$O. * ' * * Normal rubjects(Lambert, van kssel, 8 Leplat, 1447)

rn Fanconi syndrome before treatment 8 during

x Fanconi syndrome after trwtment

0 0 9. 4 o Normal subjects (Mllne.1951)

.* phosphate infusion

4 8 I2 16 SERUM INORGANlC PHOSPHORUS (MG. PER 100 ML.)

FIG. 1. The relationship of the phosphate/creatinine clearance ratio to the serum phosphate level as derived from phosphate infusions by Milne, Stanbury

and Thompson (1952), and Lambert, van Kessel and Leplat (1947).

We now turn to our own observations, We have measured the phosphate/creatinine clearance ratios in a number of dif- ferent conditions, and in Fig. 3 we show the results obtained in 16 cases of osteoporosis, 10 cases of osteomalacia, 4 mild

224 B. E. C. NORDIN AND RUSSELL FRASER

cases of hypoparathyroidism, 4 cases of parathyroid tumour and 24 normal subjects. It will be seen that the data on our normal and osteoporotic subjects (with one exception) lie within, or on the edge of, the 95 per cent limits which we have calculated from Alilne, Stanbury and Thompson’s data.

S E R U M INORGANIC PHOSPHORUS (mg per looml.)

FIG. 2. ,411 data from Fig. 1 up to a serum phosphate level of 8 mg./100 ml. The mean regression line and the 95 per cent limits have been calculated by the present authors.

All the cases of osteomalacia and primary hyperparathyroid- ism have higher clearance ratios with low serum phosphate levels, and 3 of the hypoparathyroid cases have abnormally low clearance ratios with high serum phosphate levels.

Our next step has been to calculate the departure of each point’s C,/C,, from the normal mean line by subtracting the

INDIRECT ASSESSMENT OF PARATHYROID FUNCTION 225

clearance ratio that would be expected normally for the serum phosphate level from the observed value. This can be done

I

4 1

S E R U M P mg/IOOn?l

FIG. 3. Relationship of the phosphat,e/crentinine clearance ratio to the serum phosphate level in normal subjects and in patients with osteoporosis, osteomalacia, hyperpara thyroidism and hypo- parathyroidism (authors' cases). Regression line

and 95 per cent limits as in Fig. 2.

with the help of the regression equation, since in normal people the following equation should hold :

P 20

c,/c,, - - = - 0 * 0 5 & 0 .12

P 20

or C,/C,, - - + 0 . 0 5 = 0 f 0 .12

When this is done, the results shown in Fig. 4 are obtained. We have called this measure of the abnormality of the C,/C,,

BONE 9

226 B. E. C. NORDIN AND RUSSELL FRASER

(and so of renal phosphate excretion) the “ Phosphate Excre- tion Index” (P.E.I.). A figure above + 0 .12 or below - 0 . 1 2 should then signify an abnormal phosphate/creatinine clear- ance ratio, irrespective of the serum phosphate level, and it will be seen that this single figure separates patients with abnormal parathyroid function from normal subjects. The

P ~ R ~ l k l f l O l D 1”r:ilR . . -

H O R M A L

M I S C E L L A N t O U S

0 Sl f O P O R O S I S

HIP 0 P A R A T H I ‘ R O I D

. FIG. 4. The mean expected phosphate/creatinine clearance ratio for the blood level is defined as zero. The normal range embraces two standard deviations

above and below zero (i.e. - 0.12 to + 0.12).

10 cases of osteomalacia (with presumed secondary hyper- parathyroidism) and the 4 of parathyroid tumour all lie above the normal range, and 3 of the hypoparathyroid cases fall below it. Most of the “miscellaneous” cases fall within the normal range, but there are 9 above it, made up as follows:

Cushing’s syndrome 4

? Parathyroid tumour 1 M yxoedema 1

Renal stone 2 Vitamin D intoxication 1

IKDIRECT A 4 ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ OF PARATHYROID FUNCTION 227

The high values in Cushing’s syndrome are of interest and suggest that the low serum phosphate of this condition is associated with impaired tubular reabsorption of phosphorus. Could this be due t o potassium depletion?

Now I wish to turn aside for a moment to remind you of the effect of intravenous calcium on phosphate excretion (Howard,

The effect of L k! Ccdciurn on:

1-

0

Serum Phosphate (mean t se.)

I I I I Osteomalacia (3

3~ Osteoporosis 00) -Norm1 a

I I Jh

Hopkins and Connor, 1953). We have already published our findings on this subject (Nordin and Fraser, 1954) and Figs. 5a and 5b are taken from our previous paper. They show that a 4-hour infusion of calcium gluconate (15 mg. Ca per kg. body weight) is followed by an immediate rise in serum phosphate and a progressive fall in C,/C,,, the latter reaching its lowest point about 12 hours after the commencement of the infusion. We have already reported our reasons for believ- ing that these reverse changes in serum and urine phosphate

228 B. E. c. NORDIN .4ND RUSSELL F R A S E R

are due to suppression of the parathyroid glands by the ele- vated serum calcium. As Dr. Howard has just stated, there is a good deal of evidence that the activity of the parathyroid glands is controlled by the concentration of calcium in the body fluids; presumably i t is, in fact, the concentration of

The effect of I c! Cofcium on.

Phosphate . Creatinine clearance ratio

(mean i s e 1

ma Osteomalacia 0) = Osteoporosis 09

Normal (19

4 0 4 8 12 16 20 HOURS

FIG. 5b.

ionized calcium which is the important factor. Since elevation of the serum calcium apparently leads to suppression of para- thormone secretion, we thought that reduction of ionized calcium by infusion of sodium citrate might have the opposite effect. Other workers (Stewart and Bowen, 1952) have already shown that the injection of oxalate and citrate salts into

INDIRECT ASSESSMEKT OF PARATHYROID FUXCTION 229

4

J

L " J

2 2

I

......

C O N T R O L D A Y

FIG. 8. Two 8-hour infusions of sodium citrate in normal subjects. In each case the serum phosphate, urine phosphate output, and phosphate/creatinine clearance ratio on the control day and the infusion day are shown. The dif- ferences in serum level may not be significant. but infusion of citrate is followed by a marked rise in phosphate output per hour and in the phosphate/

creatinine clearance ratio.

animals leads to a rise in serum calcium which is believed to be due to stimulation of the parathyroids, but t o the best. of o w

230 B. E. C. NORDIN AND RTJSSELL FRASER

knowledge no similar observations have been made in man. Milne (1951) observed a rise in urine phosphate after infusion of sodium citrate, but he did not attribute i t to parathyroid gland stimulation.

CHANGES IN “PHOSPHATE EXCRETION INDEX’

t‘) -

t . 2 5 -

+ .2 -

$ + I S -

L

Y = t . 1 -

Y b 5 t . 0 5 -

E : .o - E 2 I - . 0 1 -

- .I - - 4 5 -

-.zo -

J7.5 C. IU 8 RQURS.

- 4 s J I I I I I I I 1

0 4 B I2 16 20 1 4 nouns

FIG. 7. A comparison of the effect of calcium gluconate and sodium citrate infusions on the “phosphate excretion index”. By contrast, the diurnal variations in 3 normal subjects do not extend outside the

normal range.

We started by infusing fairly small amounts of sodium citrate (12 .5 g. in 4 hours) and observed a slight phosphate- diuretic effect. As the dose was increased, the effect became more marked, and the result of two 8-hour infusions (37 * 6 and 50 g. respectively in 8 hours) are shown in Fig. 6. In each case,

INDIRECT ASSESSMENT OF PARATHYROID FUNCTION 231

the serum phosphate, the urine phosphate in mg. per hour, and the C,/C,, are shown. The slight differences between the serum phosphate levels on the control days and on the citrate days are probably not significant, but in each subject there was a substantial rise in total phosphate output and in C,/C,,

I r I I I I 1 I I I I , I

FIG. 8. The effect of calcium gluconate and sodium citrate infusions in a patient with hypoparathyroidism. hleither infusion was associated with the reverse

changes in serum and urine phosphate which occurred in normal subjects.

0 4 b I2 16 0 4 b I2 I6 H o u m s wouns

following the citrate infusion. A little tingling around the mouth was the only sign that the ionized calcium level was in fact being reduced.

In Fig. 7 we have compared the effect of calcium gluconate and sodium citrate infusions on the Phosphate Excretion Index with the diurnal changes seen in three normal subjects without any infusion. This method of presentation shows

232 B. E. C. NORDIN ASD RUSSELL FRASER

more clearly the significance of the changes that are occurring, and demonstrates that calcium infusion reduces the C,/C,, to definitely subnormal levels, while citrate infusion raises it into the hyperparathyroid range. By contrast, the diurnal variations in C,/C,, and serum phosphate level in 3 normal subjects are related in such a way that the P.E.I. remains within the normal range throughout.

It is difficult t o prove that the effects we have described are due to changes in parathyroid activity, but their failure to occur in hypoparathyroidism offers some support for this hypothesis. Fig. 8 shows the effect of calcium gluconnte and sodium citrate infusions on the serum phosphate, C,/C,, and P.E.I. in a case of postoperative hypoparathyroidism. The case was a mild one and was fairly well controlled on calciferol when the citrate infusion was performed. However, i t is clear that no significant change occurred in the tubular reabsorption of phosphate after either calcium or citrate infusion.

I n conclusion, our observations suggest that abnormalities in renal tubular reabsorption of phosphate, including varia- tions in parathyroid activity, may conveniently be expressed by the Phosphate Excretion Index.

REFERENCES HOWARD, J. E., HOPKINS, T. R., and CONNOR, T. R. (1953). J . din.

Endocrin., 13, 1. LAXBERT, P. P., VAN KESSEL, E., and LEPLAT, C. (1947). Actrr med.

scnnd., 128, 386. RIILNE, RI. D. (1951). The Action of the Parathyroid Hormone, J1.D.

Thesis, Manchester. ~ I ILNE, M. D., STAXBURY, S. W., and THOMPSOX, A. E. (1952). Quart. J .

Med., 21, 61. SORDIN, B. E. C., and FRASER, R. (1954). Clirz. Sci., 13, 477. STEWART, G. S., and BOWEN, H. F. (1952). EndocrinoZogy, 51, 80.

DISCUSSION Dimn: At Stanmore we calculated our phosphate clearances by

reference to a 24-hour period, when a patient was in the metabolic ward on an absolutely constant intake so that all the tissues, blood and urine were in equilibrium, as it were. I felt that in the clearances of

DISCUSSION 233

phosphate, described by Dr. Nordin, that were calculated by intraven- ous infusion when the blood was brought temporarily to a higher level than the tissues, the phosphate then went two ways: some went out cleared by the kidney and some went into the water of the other tissues. The picture was not quite so clear as if you calculated the clearance with everything absolutely constant on a fixed diet. We have had patients who have been for experimental purposes on widely varying phosphate intakes, on which the blood levels of phosphate were not materially affected, and yet there were very wide variations indeed in the phosphate clearances when calculated from 24-hour urine phosphate divided by the blood level, giving ml./min. of phosphate cleared. By doubling up the phosphate intake, for instance, you could get an apparent doubling up of the phosphate clearance and yet the blood level remained constant. The actual glomerular filtration rate was checked now and again by inulin clearances, not by creatinine clearances. It is very difficult to estimate plasma creatinine a t the ordinary endogenous level. The uptake is so low that it is difficult to get any accurate creatinine clear- ance. Have you measured plasma creatinine in your calculations on clearances ?

Nordin : This is a basic point: I do not feel that you can call a 24-hour collection, with the blood taken some time during 24 hours, a clearance. The whole essence of the clearance phenomenon is that you take a period of time, you take your blood in the middle of that, and you assume that your blood level is not changing. We know that there are big diurnal variations in the phosphate and I consider that even &hour clearance is a bit too long to be quite right. We now do the base-line clearance in the morning, which is our standard. We do that on a 2-hour basis and I think 2 hours is about as long as you can reasonably do a clearance on a blood level which you know is fluctuating. So I would be reluctant to call those data of yours clearances. We have looked into the question of the effect of diet, and have given a normal subject varying amounts of P in the diet. The serum phosphate level varied from 2 .5 to 4.3 and as it did so the C,/C,, went up as predicted so that all the observations were in the normal range of Milne, Stanbury and Thompson. You cannot talk of a phosphate clearance without simultaneously referring to the serum phosphate level. I do not think that one observation of serum phosphate in 24 hours can meet that point.

Dizon: If you give diets high in phosphate you will not materially affect the serum phosphate levels, but you will shoot out enormously increased amounts of phosphate. That means your phosphate clearances have gone up.

Nordin: The point is that you do change the serum phosphate level. Dizon: Not SG markedly as the increase in urine phosphate. You

might alter it by 10 or 20 per cent, but not more. Dent: That depends on the slope of his curve. Nordin: As your blood level goes up you divide by that blood level,

but phosphate excretion goes up so much that the clearance rises too. As far as the blood creatinine is concerned I agree. I think it is a very tricky thing and I wish we did not have to estimate it, but methods are

234 DISCCSSION getting better and I think we are accurate within 10 per cent in our blood creatinine levels. We have done a lot of duplicates and triplicates a t dif- ferent times of the day and they certainly are within 10 per cent if done by Roscoe's method. There are other methods which we hope to use. But I think even if you take into account the errors in the blood creatin- ine level, the differences that I have shown you remain highly significant.

SERUM PHOSPHORUS mg%

FIG. 1 (Nassim).

Figs. 1, 2 and 3 show studies of phosphate clearance in 3 women before and after treatment with stilboestrol.

Points to be noted are that on each occasion there has been no change in glomerular infiltration, and that after treatment the slope has been shifted to the left, showing lowering of tubular phosphate re-absorption.

In Fig. 3 the clearance was done on stilboestrol first, and a fortnight after withdrawal, and therefore the oestrogen effect is probably persisting.

Nassim: Were the osteoporotics which you showed in Fig. 5 post- menopausal women?

Nordin: Most of them were. There are four cases of what we call preg- nancy osteoporosis, and Dr. Dent calls idiopathic osteoporosis, included in that.

Nassim: It seems to me that those levels were getting on the lower side of normal, towards the hypoparathyroid level.

DISCUSSION 235

Nordin: No, it might have been so, but so far it has not turned out like that. They are all over the normal range, and the mean and standard deviation of the osteoporotics and the normals, calculated independently, are the same.

A’assim: My colleagues, Drs. P. D. Saville and Lily Watling, and I have studied this. As is well recognized, the serum phosphate in post- menopausal osteoporosis tends to be high normal. This, we feel, may be due to an anterior pituitary effect. When patients are treated with stilboestrol the serum phosphate tends to drop. Phosphate clearances

TREATED UNTREATED

G F R 96 94 ml. p.. itnut*

Ihronticol 3 . 5 4 . 9 mq 2 TmP 3.4 4 . 6 m j p r m i u t e T h n r h a l d

SERUM PHOSPHORUS mg % FIG. 2 (Kassim).

have been performed on three women, using the technique of Anderson (1955, J . Physiol., 130,268). All three studies show clearly the increased phosphate clearance after administration of stilboestrol (Figs. 1-3).

Nordin: It is not easy to convert these figures straight o\-er; this is phosphate excretion in mg./min. It requires a bit of mathematics to convert that to a clearance ratio. I think there is obviously a slight change in tubular reabsorption but I suspect that the biggest change is in the blood level and that the consequent change in tubular reabsorp- tion leaves them both still within the normal range. Do you think that is possible ?

Delzt: Yes , there is such a wide range.

236 DISCUSSION

Nassim: There is no doubt about the fact that phosphate clearance is altered with stilboestrol.

Pollis: Dr. Kordin, arc your cases of osteomalacia histological or radiological ?

Nordin: The osteomalacias are absolutely water-tight. The osteopo- roses are just what people a t the moment ordinarily call osteoporoses.

TREATED UNTREATED

G.F.R. 58 53

Theoretical 2 0 2 . 8 Threihoid

2 4 6 8 10 12

SERUM PHOSPHORUS mg.%

FIG. 3 (Nassim).

But for the purposes of this presentation here they include what Dr. Kassim calls grades I, I1 and 111. In other words, they are people whose bones look thin and who have got back-ache, and those who have cod- fish vertebrae, and those who have got crush fractures. We try to confine ourselves to the ones with cod-fish vertebrae and crush fractures.

Kodicek: Would it be possible to estimate the clearance ratio by isotopic dilution techniques, or has this been done? Another point, could you not call your index a variance?

DISCUSSION 237

Nordin: Yes, I suppose that is really a better term than the term “phosphate excretion index”. I do not know if one can calculate the clearance ratio by radio-isotopic dilution techniques.

Nicolaysen: Did you measure the citrate output in the urine? Nordin: No, we did not measure the citrate output in the urine; we

measured the blood citrate level in two of them. It went up to 12 mg. per cent in one and 17 mg. per cent in the other.

Nicolaysen: Alkali ingestion may have an influence. Nordin: I have tried bicarbonate and if bicarbonate does anything

it does the opposite; i t puts down the serum phosphate and the urine phosphate a t the same time. As far as the base is concerned our standard dose is now 500 ml. of 5 per cent sodium citrate given in four hours; that is about 100 m-equiv.

Nicolaysen: Are you sure that your creatinine clearance is not influ- enced by this difference?

Nordin: Dr. Dent raised this question on our Ca infusion work, so we did a few inulin clearances a t that time, but they did not make any very useful contribution. We have published (Nordin, B. E. C., and Fraser, R. (1954), Clin. Sci., 13, 477) the creatinine output per hour after Ca infusions and we think it is unchanged, although there is a tendency for people to put out slightly less creatinine in the night than they do in the day. I have not worked out the same thing after citrate infusions, but it would have to be a very big rise in GFR to account for this sort of rise in phosphate output.

Blaxter: If you give the kidney more phosphorylating work to do, that is if the kidney has to do more in the way of taking up inorganic phosphate for phosphorylating purposes, does that change your clearance ratio and can that account for the effect of citrate?

Nordin: Why does it have to take up more? The citrate has not put the serum phosphate level up. We are not using anything that influences the phosphate level itself. As I showed you, the serum phosphate on the citrate day is a little lower than the serum phosphate on the control day. So you would expect that the urine clearance should be lower, if any- thing, but in fact the urine clearance is always higher.

Blaxter: I was thinking of the phosphorylation in the wall of the tubule on reabsorption.

Nordin: But why should there be a change in phosphorylation if there is no change in blood level of phosphate?

Blaxter: That is a systemic blood level, isn’t i t ? It tells one little about the phosphate levels in the renal cells.

Armstrong: May I put a general question that has to do with the subject of mechanism of action of parathormone? I think we all are con- vinced that the parathyroid hormone does demonstrably have an effect directly on the bone and that it also alters phosphate reabsorption by the renal tubules. The evidence for both of these points is very clear. My question is : how is it that a single substance can affect vastly different physiological processes in ways that produce the same result, that is the mobilization of the skeletal elements? Can anyone point out to me an analogous circumstance in the case of some other substance?

238 DISCUSSION Kodicek: Vitamin D. Armstrong: How? Kodicek: Again it is rather vague, vaguer than parathormone; but

there is a suspicion that vitamin D exerts its influence not only on the intestine, but has also a local action in bone, and possibly affects the reabsorption in the kidney tubules.

Funconi: If Dr. Nordin’s paper is right we must avoid giving citrate in osteomalacia, and, on the contrary, we now give citrate in the treat- ment of osteomalacia.

Nordin: I do not believe that the oral administration of citrate causes anything like the rise in blood level which we are getting with our infusion.

Nicoluysen: I think you should differentiate very sharply between the solubilizing effect of citrate on Ca in the intestine and the postabsorp- tive effect.