The interpretation of laboratory tests in bone disease

The Interpretation of Laboratory Tests in Bone Disease OLAV L. M. BlJVOET JAAP VAN DER SLUYS VEER

Interpretation of biochemical measurements requires experience of physiological concepts and presumptions. This chapter summarises some current presumptions underlying the interpretation of laboratory investigations of disordered calcium metabolism associated with bone disease. Figure I is Fuller Albright's classic diagram of calcium metabolism (Albright, 1947) slightly modified. In his diagram the confines of the body (I) were reduced to a rectangular affair with four rudimentary appendages (2) to make it more realistic. The fluid in the body is divided into several compartments, but only the plasma (3) is represented separately Note that the calcium concentration in plasma is almost twice that of the other fluids due to the binding of calcium to plasma protein. The gastrointestinal tract (5) is represented with the direction of dietary calcium intake (6), calcium absorption (7), secretion (8) and faecal excretion (9) of calcium. The kidney (10) is depicted as a nephron with glomerular filtration (II), tubular reabsorption (12) and urinary excretion (13). The skeleton is represented by a rectangular mass (14) in the centre of the body. Bone is partly covered by a layer of osteoid where new bone is being laid down by osteoblasts (15), and there is a region (16) where bone is removed by osteoclasts during remodelling. The osteocytes in the bone may exchange calcium (17,18) by a process called osteolysis. Finally, the calcium ions in the bone exchange directly and freely with calcium ions in the extracellular fluid (19,20). There is some exchange of calcium with 'dystrophic' calcium deposits outside the bone (21). The concentration of calcium in the extracellular fluid is constant when the rates of calcium transfer to and from the body through gut and kidneys (external calcium turnover, 22) and the rates of calcium transfer to and from the bone (internal calcium turnover, 23) are in equilibrium. A homeostatic mechanism maintains the calcium concentration of the body fluids (24) within narrow limits. The arrows in Figure I represent rates and directions of transfer of calcium. The methods for measuring these various rates of calcium transfer, and their role in maintaining the homeostasis of plasma calcium concentration will be discussed. 217

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HUVOET AND JAAP V AN DER SLUYS VEER

The plasma calcium concentration is monitored by the parathyroids and, perhaps, the calcitonin-producing cells. These glands regulate the equilibrium oetween the various rates of calcium transfer. There is no similar hormonal regulation of the plasma phosphate level, which depends mainly on the kidney. The discussion of phosphate metabolism will therefore be limited to plasma phosphate concentration and the renal handling of phosphate.

r---""'T"-----------"""""1

2

3

2 x rCa]

____-+-:::----'~-----....,7 8

----"0. ~

[cal 24

2

Figure 1. Diagrammatic representation of calcium metabolism in a healthy adult person. See text for explanation. Reproduced, with permission, from Albright, F. (\ 947) Hormones and human osteogenesis, Recent Progress in Hormone Research, 1, 300. Copyright held by Academic Press, New York.

PLASMA CALCIUM AND PHOSPHATE The range of plasma calcium concentration, 9·1 to 10·7 mg/IOO ml, is small (Keating, Jones and Elveback, 1969). Calcium values may vary between laboratories (Veali, 1962) and according to the technique of extimation (MacIntyre, 1957; Zettner and Seligson, 1964). Classic estimation methods involve precipitation of calcium as the oxalate (Clark and Collip, 1925) or complexing with ethylenediaminotetraacetic acid (EDTA). Newer methods depend on emission, or absorption, spectrophotometry (MacIntyre, 1957; Zettner and Seligson, 1964). The last two methods cannot be used to evaluate the effect of EDTA on plasma calcium. A little over 50 per cent of plasma calcium is ionised (Table I); less than half of total calcium is bound to protein, and in ultrafiltrates, such as the renal glomerular filtrate, the calcium

219

THE INTERPRETATION OF LABORATORY TESTS IN BONE DISEASE

Table 1. State of Calcium in Normal Human Plasma mg/loo ml

% total

4·72 4·56 0·16 0·16 0·32 9·92

47·5 46·0 1·6 1·7 3-2 100

Free calcium ions Protein bound CaHP0 4 Calcium citrate Unidentified Total

Reproduced, with permission, from Walser, M. (1961) Ion association. VI. Associations between calcium, magnesium, inorganic phosphate, citrate and protein in normal human plasma. Journal of Clinical Investigation, 40, 723-730.

concentration is about 58 per cent of the plasma calcium concentration (Walser, 1961). Direct measurements of ultrafiltrable calcium (Toribara, Terepka and Dewey, 1957; Yeall, 1962) are too complicated to be used routinely. The ionised calcium is under hormonal control, and is physiologically the most important fraction. Methods are being developed for a direct assay of ionised calcium with a selective electrode (Ross, 1967). Protein binding of calcium varies with protein concentration and with pH. These relationships have been worked out by McLean and Hastings (1935). Serial studies or comparisons of plasma calcium concentrations are incomplete when total protein concentration is not accounted for. Plasma protein and hence plasma calcium concentration are influenced by venous stasis during venipuncture, they increase when the patient changes from supine to upright position (Smeenk, 1968) (Table 2) and they may vary during the day (Fourman and Royer, 1968). Neglect to account for this may diminish the value of sequential studies of plasma calcium. Table 2. Concentration in Plasma (± 2 s.e. mean) of Protein, Calcium and Phosphate after 30 Minutes Standing and after 45 Minutes Recumbency

No. Protein Calcium Phosphate

(g/loo ml) (mg/l00 ml) (mg/loo ml)

38 38 36

Upright 7-67 9·83 3·48

± 0·17

± 0·12

± 0·17

Recumbent 7·00 9-48 3-45

± 0·15

± 0·10

Difference 0·67 0·35

± 0·11

± 0·06

± 0·17

Reproduced, with permission, from Smeenk, D. (1968) Some aspects of hyperparathyroidism. Folia Medica Neerlandica, 11, 194-201.

Many factors influence the individual routes of calcium transfer sketched in Figure 1 and tend to disturb plasma calcium concentration, but homeostatic actions of the parathyroid hormone and calcitonin commonly override these effects. Hypercalcaemia is due either to a regulation defect of parathyroid hormone secretion, to increased renal tubular reabsorption of calcium, to increased absorption from the gut, or increased bone destruction (see below). Hypocalcaemia, when not due to a low total plasma protein, is most often caused by hypoparathyroidism, or to end-organ insensitivity to parathyroid hormone. The latter occurs in pseudohypoparathyroidism and, secondarily, in osteomalacia or chronic renal failure. Hyper- or hypo-

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parathyroid patients can be normocalcaemic. The disorders may then be discovered by repeated determinations of plasma calcium, or by challenging the homeostatic mechanisms-chlorthalidone (Seitz and Jaworski, 1964), low P-diets (Dent, 1962), or EDTA infusions (Fourman and Royer, 1968)-and by simultaneous determination of plasma parathyroid hormone levels. In contrast with calcium, most of the phosphate in plasma is in a diffusible form (Table 3) and only about 12 per cent is protein bound. Yet phosphate concentration in ultrafiltrates of plasma, such as the glomerular filtrate, Table 3. State of Phosphate in Normal Human Plasma

Free HPO~Free HzPO;j Protein bound NaHPO;j CaHP04 MgH P04 Total

mg/100 ml

% total

1·55 0·34 0·43 1·02 0·12 0·10 3'56

43 10 12 29 3 3 100

Reproduced, with permission, from Walser, M. (1961) Ion association. VI. Associations between calcium, magnesium, inorganic phosphate, citrate and protein in normal human plasma. Journal of Clinical Investigation, 40, 723-730.

equals the concentration of phosphate actually measured in the plasma; this is because current techniques of estimation do not take into account the volume taken up by plasma proteins and because the distribution of phosphate ions between the two sides of an ultrafiltering membrane is influenced by the charge of the proteins remaining at one side (the Donnan equilibrium) (Walser, 1961). Erythrocytes contain phosphate, and failure to centrifuge blood immediately after sampling may give erroneous plasma phosphate values. There is considerable variation in plasma phosphate (Keating et ai, 1969). It varies from 2·4 to 4'4 mgjlOO ml in healthy adults. The concentration is high in childhood and adolescence and it has a circadian rhythm (Wesson, 1964) which is at least in part dependent on phosphate intake. When comparing groups of people, age, phosphate intake and the time of sampling should be taken into account (Greenberg, Winters and Graham, 1960), as these all cause variations in plasma phosphate. Many factors may influence the plasma phosphate concentration (Danowski, 1962). A transient reduction occurs after glucose feeding. Plasma phosphate may be low in renal tubular defects, in potassium depletion, in phosphate depletion, hyperparathyroidism, and hypercorticism. It is frequently high in hypoparathyroidism, thyrotoxicosis, growth hormone excess, hypocorticism and in some patients with Paget's disease (Fourman and Royer, 1968). The relation between plasma phosphate and glomerular filtration will be discussed later The calcium X phosphate product has been invented to account for physiological processes in terms of calcium-phosphate precipitation. This product is not physiologically regulated (Neuman and Neuman, 1958). Because of the much greater variation of plasma phosphate, it mainly depends upon phosphate concentration when plasma calcium is not grossly abnormal.

221

THE INTERPRETATION OF LABORATORY TESTS IN BONE DISEASE

HOMEOSTASIS AND THE KIDNEY Three aspects of renal function will be discussed. I. The excretion rate (U V; mass/time). In a steady state the excretion rate equals 'extrarenal load', that is the net rate at which a substance enters the extracellular compartment at sites other than the kidney. In a transient state a sudden change of UV may be due to a change in renal handling of a substance, or due to a change in plasma concentration or to both. 2. The rate of tubular reabsorption (T; mass/time) of a substance taken in relation to the net rate of fluid reabsorbed from the glomerular filtrate (vol./ time) determines the concentration at which a substance is reabsorbed. Provided that no tubular secretion occurs, this may contribute to the setting of the plasma concentration of a substance. 3. Glomerular filtration rate (G. F. R.; vol.jtime) multiplied by the concentration of a substance in the glomerular filtrate (rnass/vol.), determines the filtered load (L; mass/time). PHOSPHATE

Excretion rate The amount of phosphate excreted is equal to the amount of phosphate a bsorbed from the diet. The average diet contains about 1500 mg phosphate per day and the average 24-hour renal excretion of phosphate is about 600 mg per day; variations of intake and urinary output are closely related (Nordin and Smith, 1965). Phosphate excretion has a circadian rhythm; part of this is due to diurnal variation in feeding (Dossetor, Gorman and Beck, 1963). Sequential studies of phosphate excretion should be accompanied by control studies over comparable periods. The most common causes of a low phosphate excretion are phosphate-poor diets, such as low protein or low calcium diets, or administration of aluminium hydroxide gels which bind phosphate in the gut (Fauley, 1941). Hyperphosphaturia may result from high phosphate intake or breakdown of bone, and in a steady state is never due to decreased renal tubular reabsorption of phosphate. It is misleading to characterise as phosphaturic (Chambers et al, 1956) the renal effects of a hormone like parathyroid hormone which merely sets tubular phosphate reabsorption at a lower level (Bijvoet, 1969). It is only a sudden increase of the circulating level of the hormone which will cause a transient increase of phosphate excretion, and excretion rate will return to normal when a new steady state is established.

Tubular reabsorption of phosphate The rate at which phosphate is filtered through renal glomeruli (filtered load,

L; mass/time) equals the product of G.F.R. and plasma phosphate concentration ([P]; mass/vol.) (Bijvoet, 1969). Most of the filtered phosphate is reabsorbed in the proximal tubules. Urinary phosphate excretion (UP04 V; mass/time) is the non-reabsorbed phosphate, since in man there is no evidence for tubular secretion of phosphate (Bijvoet, 1967). UP04V

=

Lp04 -

Tpo4 or UP04V

= G.F.R.

X [P] -

Tp04

(I)

When [P] increases, the phosphate concentration in the filtrate increases.

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As a result the phosphate reabsorption from the glomerular filtrate increases (Figure 2). There is an upper-limit, the 'tubular maximum' (Tmpo4; mass/time) to the rate at which phosphate can be reabsorbed from the glomerular filtrate (Schiess et al, 1948). When Tmpo4 is reached, any additional increase of the filtered load is entirely excreted. The point at which the regression line in Figure 2 cuts the abscissa is called the 'theoretical renal phosphate threshold' (Brain, Kay and Marshall, 1928; Cushny, 1917). It equals Tmpo4/G.F.R. (mass/vol.), the maximum rate at which phosphate can be reabsorbed from a unit volume of glomerular filtrate.Tmp04/G.F.R. measures the setting of tubular phosphate reabsorption (Bijvoet, 1969). This can be easily observed in Figure 2. When [P] is small in relation to the 'threshold' (Tm/G.F.R.), the excretion rate is small. When [P] is large in relation to Tm/G.F.R., excretion rate increases in proportion to [Pl. The tubular reabsorption of phosphate depends on Tm/G.F.R. as the relative magnitude of [P] in relation to Tm/ 12

10

•

2

o I<-_-"'~f--'---'-----'-----'---'-'

o

2

4 Plasma Tmpo 4 G.F.R.

6

8

ro

12

[p] (mg/100ml)

Figure 2. The relation between the urinary excretion rate of phosphate (UP04 V; mgjmin) and the plasma phosphate ([P]; mgjlOO ml) in a healthy person when fasting (open circle) and during an infusion of phosphate (closed circles). Note that plasma [PI equals the phosphate concentration in the renal glomerular filtrate. The open squares show the relation between urinary excretion rate and plasma concentration of inulin when inulin was infused simultaneously (the inulin results are divided by 10). The slope of the line through the infusion data for phosphate is the same as the slope of the line through the inulin data and is therefore the glomerular filtration rate (ml x lO-Zjmin). The vertical distance between the two straight lines, or the negative intercept on the ordinate of the extrapolated straight line through the closed circles, is the maximum rate of tubular reabsorption of phosphate (Tm;mgjmin). The intercept of the line through the closed circles with the abscissa is the maximum tubular reabsorption of phosphate per 100 ml of glomerular filtrate (TmjG.F.R.; mgjlOO ml) which has also been called the 'theoretical renal phosphate threshold'. Reproduced, with permission, from Bijvoet, O.L.M. (1969) Relation of plasma phosphate concentration to renal tubular reabsorption of phosphate. Clinical Science. 37, 23-36.

THE INTERPRETATION OF LABORATORY TESTS IN BONE DISEASE

223

G.F.R. determines what proportion of filtered load is reabsorbed (TIL), even when the absolute rate of reabsorption is not maximal. This relation has been worked out in Figure 3 for a wide range of values ofTm/G. F. R. in 100 persons when fasting and when infused with phosphate (Bijvoet and Morgan, 1971). MEASUREMENT. The reabsorbed fraction of the filtered load (T/L) is often called T.R.P. But others have used T. R.P. for T (mass/time) (Bernstein, Yamahiro and Reynolds, 1965). It is the complement of UV/L, the excreted fraction of the load. UV/L equals the ratio of phosphate clearance (CP04; vol.rtime) to creatinine clearance (Ccreat = G.F.R.; vol.jtime). Thus, (2) and T/L = T.R.P. = I - (Cpo4/Ccreat) (3) This ratio can be easily obtained from simultaneous measurements of phosphate and creatinine concentrations in plasma and urine over a short untimed collection period. The ratio CP04/Ccreat can be calculated as follows:

UP04V

[creat]

--x--[P]

UcreatV

Since the term V is common in both clearances it can be eliminated which makes accurate timing and volume measurement superfluous. The formula becomes UP04 X [creat] Cpo4/Ccreat = - - - - - -

Ucreat X [P] Cp04/Ccreat is equal to the excreted fraction of filtered phosphate load because:

[P] x G.F.R. Lpo4 assuming that Cere at equals G.F.R. Because Tmp04/G.F.R. measures the setting of phosphate reabsorption, indices of phosphate reabsorption should always be related to Tm/G.F.R. (Bijvoet, Morgan and Fourman, 1969). Direct measurement ofTm/G.F.R. by phosphate infusion (Figure 2) is difficult and time consuming and cannot easily be repeated. A much simpler method is to use Figure 3 as a nomogram to obtain Tm/G.F.R. from simultaneous measurement of T/L = 1-(Cpo4/ Ccreat) and [P] (Bijvoet and Morgan, 1971). The figure can be used to derive Tm/G.F.R. x I/[P], which is the abscissa, from the measurement of T/L (ordinate). Multiplying the abscissa by [P] gives Tm/G.F.R. Note that when Upo4V/L > 0·20 (T/L < 0'20) then TIL equals Tm/L and Tm/G.F.R. simply equals [P] x TIL. The figure shows that T/L will decrease and thus that UV/L will increase as [P] increases, for any given fixed value of Tm/G'F, R. This relation between UP04 V/L (which equals Cpo4/Ccreat) and [P] is given in Figure 4 for a series

OLAV L. M. BlJVOET AND JAAP VAN DER SLUYS VEER

224

of increasing values of phosphate reabsorption (Bijvoet et ai, 1969). The figure relates most clinical indices of phosphate reabsorption, including the phosphate excretion index (PEl) which was an early attempt to approximate the normal relation between Upo4VjL and [P] by a straight line (Nordin and Fraser, 1956). MEANING. The setting of phosphate reabsorption (TmjG.F.R.) influences the setting of the plasma phosphate [P] in a fasting state in a predictable manner. I t determines most of its variation and the variation of [P] is only to a small extent due to either variation of extrarenal load (= UV) or of G.F.R., when G.F.R. varies within the normal range (Bijvoet, 1969). The normal range of variation of TmjG.F.R. is between 2·5 and 4·2 mgjIOO ml (Bijvoet and M organ, 197 I; Stamp and Stacey, 1970). Phosphate reabsorption (Tmj G.F.R.) is influenced by parathyroid hormone, it is decreased in hyperparathyroidism and increased in hypoparathyroidism. It is also decreased by calcitonin, oestrogens, adrenal steroids and potassium depletion. It is increased by growth hormone, in thyrotoxicosis, in patients with severe Paget's disease or after prolonged heparin treatment. Vitamin 0 may .60

.40

!

uv

L

L

_ _ .30

.70

'"~

Cl..

.20~

C\: .80 f-..

~

d.90

1.00 1.50

.10

0 1.40

1.30

1.20

1.10 1.00 Tmpo. x --.L [pJ G.F.R.

.90

.80

.70

.60 Tm L

Figure 3. The relation between fractional reabsorption of filtered phosphate (TIL) and the ratio of the renal phosphate threshold to plasma phosphate «Tm/G.F.R.): [PI = TmIL) in 100 persons when fasting and during an infusion of phosphate. The values of TIL have been grouped for successiveintervals of 0·1 of TmIL. The figure can be used as a nomogram for the estimation of Tm/G.F.R. from simultaneous measurements of Cp04/Ccreat and [Pl. Reproduced with permission from Bijvoet, O.L.M. and Morgan, D. B. (1971) The tubular reabsorption of phosphate in man. In Phosphate et Metabolisme Phosphocalcique. Regulation Normale et Aspects Physlopathologiques, ed. Hioco, D. J. Paris: Sandoz.

225

THE INTERPRETATION OF LABORATORY TESTS IN BONE DISEASE

decrease it and calcium infusion may increase it. There are also congenital and acquired defects of tubular integrity with impaired phosphate reabsorption (renal rickets and osteomalacia and many patients with recurrent renal stones). Phosphate reabsorption varies also with age and is increased in children and adolescents (Bijvoet, 1967; Bijvoet et ai, 1964, 1969). Phosphate reabsorption, thus does not reflect parathormone activity solely.

Glomerular filtration rate and [P] A consideration of the effect of G. F. R. on [P] is particularly relevant because of the present concern with disorders of calcium and phosphate metabolism in chronic renal failure. The function given in Figure 3 allows prediction of the relation between [P] and G.F.R. when TmjG.f.R. is constant at the average normal level and UP04 V is allowed to vary through the normal range (Bijvoet et aI, 1964). The predicted relation is shown in Figure 5 and is compared with actual observations of Kleeman et al (1970). There is a 0 . 5 0 , . . . - - - - - - - - - - - - - - , . . . - - - - - - - - , 0.50 UI'O.Y

T

1.6

L

L

0.40

~

t 0.30

0.70

~

~

~

~ 0.20

0.80

f...:

0.10

o

""'-_ _1....-_-'-_ _-'-_----'

2

3

4

5

6

1.00

7

[pJ (mg/100 ml) Figure 4. The relation between CP04/Ccreat (= UV/L) and plasma phosphate ([PJ; mg/ 100 ml) in 100 persons grouped according to Tm/G.F.R. calculated from the relation between UV/L and (Tm/G.F.R.}/[PJ shown in Figure 3. The closed circles are the average values for the groups when fasting, and the open circles when UP04 V was 1 mg/rnin. The dotted lines show the 95 per cent range of values in healthy adult persons of Tm/G.F.R. (2'5 to 4'2 mg/loo ml) and UP04 V/G.F.R. (0'14 to 0'57 mg/loo ml). The hatched area indicates the normal range of the phosphate excretion index, a linear approximation of the relation between CP04/Ccreat and [PJ in healthy adults as given by Nordin and Fraser (1960). Reproduced, with permission, from Bijvoet, O.L.M., Morgan, D. B. and Fourman (1969) The assessment of phosphate reabsorption. Clinica Chimica Acta, 26, 15-24.

curvilinear increase in [P] with decreasing G. F. R. The same variation in UP04 V leads to a much greater variation in [P] when G. F. R. is low than when G.F.R. is normal. This also explains the striking effect on [P] ofa phosphatepoor diet combined with aluminium hydroxide in patients with chronic renal failure.

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BIJVOET AND JAAP V AN DER SLUYS VEER

CALCIUM

Excretion rate Most persons excrete less than 400 mg of calcium per 24 hours. Twenty per cent of this is ionised, the remainder is complexed (Fourman and Royer, 1968). The correlation between intake and urinary excretion of calcium is considerably less than that for phosphate (Nordin and Smith, 1965). In a steady state the rate at which calcium is excreted by the kidney is equal to the net rate at which calci um enters the extracellular space at sites other than the kidney, be it from gut or from bone or both. A high or low rate of excretion is then due to a high or low net input from bone or gut or both. It is possible that the cause of this abnormal input is still in the kidney: the hyperabsorption in some patients with idiopathic hypercalciuria, or the low net calcium absorption from the gut in patients with the nephrotic syndrome could be adaptations to a primary renal abnormality. But it is also possible that the cause of this high input is outside the kidney. Absorption of calcium in the gut is an actively regulated process. Increased absorption may explain hypercalciuria in sarcoidosis, vitamin 0 intoxication, in patients with idiopathic hypercalciuria and in growth hormone excess. Decreased absorption explains hypocalciuria in vitamin 0 deficiency and partly in uraemia. Primary increase of calcium release from the bone occurs in myelomatosis, in metabolic acidosis and thyrotoxicosis or in prolonged bedrest (Fourman and Royer, 1968).

li ii o

14 ~

~

~

~ ~

Set condition: Tmpo.!
12

,01

0" •

ii~ ~t:

10

.'or

~,~

oo,t}

8

6

t

~

:0:

~p

1 ~ ~

;

0

"

4

/y~~10 .:t'-~

• • ----- ... - ....""""' .. ,,; ..

q:

__

0- __

~::~~:::~:~ ~::-~~-/~

0 0

2

o

L.-_~_.L-_.L-_..l....-_..l....-----J

120

100

80 60 40 20
0

Figure 5. The continuous and interrupted lines represent the predicted relation between plasma phosphate (lPJ; mgjlOO ml) and glomerular filtration rate (G.F.R.; mljmin) when tubular phosphate resorption (TmjG.F.R.) remains constant at a normal value of 3·2 mgj100 ml and the extrarenal load (UP04 V; mgjmin) is allowed to vary through its normal range in healthy persons (0'14 to 0'57 mgjmin). The closed circles represent observations in normal persons and patients with chronic renal disease. (Taken from the data of Kleeman et al, 1970). Reproduced, with permission, from Bijvoet, O.L.M. and Morgan, D. B. (1971) The tubular reabsorption of phosphate in man. In Phosphate et Metabolisme Phosphocalcique. Regulation Normale et Aspects Physiopathologiques, ed. Hioco, D. J. Paris: Sandoz.

THE INTERPRETATION OF LABORATORY TESTS IN BONE DISEASE

227

Tubular reabsorption of calcium Calcium concentration in the glomerular ultrafiltrate is about 58 per cent of plasma calcium (Walser, 1961). The filtered load at the level of the renal glomeruli is about 9000 mg per day, of which 8600 mg are reabsorbed at various sites in the proximal and distal tubules of the kidney (Epstein, 1960; Fourman and Royer, 1968). It has been suggested that if calcium concentration in the glomerular filtrate is increased by intravenous infusion of calcium, about 51 per cent of any increase in filtered load is excreted into the urine (Peacock and Nordin, 1968; Peacock, Robertson and Nordin, 1969). When one increases the plasma calcium concentration and therefore the concentration of calcium in the glomerular filtrate, by administering an intravenous infusion of calcium, a constant fraction of any increase in filtered load is excreted when the plasma calcium exceeds a certain value (Kleeman et aI, 1961; Peacock and Nordin, 1968; Peacock et aI, 1969). This value can be calIed a calcium 'threshold' (Figure 6a). Tubular reabsorption of calcium is unlike phosphate reabsorption because the kidney excretes alI the increase in phosphate load above a given value but only a constant fraction of the increase of calcium load above a given value. Tubular reabsorption of calcium is more complex and behaves as if composed of two parts, one part concentration dependent (setting the fraction excreted) and one Tm limited (setting the threshold). Peacock et al (1969) proposed to correct for variation between persons in glomerular filtration rate by expressing both excretion rate (UCaV) and filtered load (the product of G.F.R. and calcium concentration in the filtrate) per unit volume of G.F.R. They thus obtained a graph relating UCa V/G. F.R. to calcium concentration. The relation between UCaV/G.F.R. and [Cal in healthy persons (Figure 6a) in their data is: UCaV/G.F.R.

= 0·30 ([Ca] - 9'5)

(4)

where 9·5 is the calcium 'threshold', and 0·30 is the product of the ultrafiltrable fraction of total calcium (0'58) and the average fraction of the increase of filtered load that is excreted (0'51). MEANING. Equation (4) can be rewritten as: [Cal

=

9·5

+ 0·33

UCaV/G.F.R.

(5)

Assuming a normal excretion rate of 200 mg/24 hr and a G.F.R. of 120 mljmin, UCaVjG.F.R. would be in the order of 0·12 mg/lOO m\. A doubling of the extrarenal load (which equals UCa V) would increase [Cal by only 0·33 X 0,12, which is 0·04 mg/lOO ml, when a new steady state is established. It is therefore possible that the average level at which plasma calcium is maintained in a healthy person depends mainly on the kidney (Nordin and Peacock, 1969; Talmage, 1956). Similarly, the low plasma calcium of hypoparathyroidism or the increased plasma calcium in hyperparathyroidism may be set by the kidney (Figure 6b) (N ordin and Peacock, 1969; Talmage, 1956). It is, however, somewhat early to conclude that the constancy of plasma calcium at an average level set by the kidney is maintained solely through the kidney. It might welI be that even a variation of [Cal of 0·04 mg/lOO ml affects parathyroid hormone secretion and leads to adaptive changes of

228

L. M.

OLAV

BIJVOET AND hAP V AN DER SLUYS VEER

2.0.--------------------.r-~

a

1.8 ~

~

1.6

~

~1.4

'-t",1.2 ~

~1.0 ~

~(J .8

~

~ ~

.6 .4

~ ~ .2 ~ ~

O+-....,...-....--....,...-~

'R1.6.--------------- --r--,--,-, ~

b

~ 1.4

"~ 1.2

o Hypoporathyroidism (Fasting") • Hyperparathyroidism (Fasting,,)

~

~

~ 1.0

~~ ~

~ .~

.8 .6

o

-\2 ~ .4 .2

o o

0

00

o

0

0

.... :

0 0

00

o

...

0

O+--,.........,.-"""'T""-...,..-.......'r-.....,,....-....:,.-.......-~-,.....~ 6

8

10 12 14 16 Total Plasma Calcium (mg/100 ml)

Figure 6a. The relation between the urinary excretion of calcium per 100 ml of glomerular filtrate (UCa VICereat) and the total plasma calcium in healthy persons when fasting (open circles) and during calcium loading (closed circles), the interrupted line represents a regression line through the closed circles. The continuous lines are drawn by hand to enclose a 'normal range'. Figure 6b. The relation between urine and plasma calcium in patients with hypoparathyroidism (open circles) and hyperparathyroidism (closed circles) when fasting (dash through the symbol) and when loaded with calcium. Normal limits as in Figure 6a. (After Peacock, Robertson and Nordin, 1969.)

calcium transfer outside the kidney. Phang et al (1969) found that changes of the rate of calcium absorption in the gut produced by changing the calcium content of the diet were only partly equalled by changes in renal excretion rate; they were further compensated by relative changes between the rates of gain and loss of calcium in the skeleton. This may explain why the correlation between calcium intake and renal calcium excretion is poor (Nordin and Smith, 1965).

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229

It is confusing to attribute the sustained lowering of urinary calcium excretion produced by thiazides (Seitz and Jaworski, 1964), exclusively to an effect on tubular reabsorption (Nordin and Peacock, 1969). If this were so, they would raise plasma calcium and the original excretion rate would be restored. The sustained decrease of excretion rate indicates (primary or secondary) extrarenal effects and it is the maintenance of plasma calcium at the normal level in the presence of a lowered excretion rate which indicates the presence of a renal effect as well. Not many agents are known that produce a sustained change of the setting of tubular calcium reabsorption, probably because their effect is immediately counteracted by the parathyroids. MEASUREMENT. The value of measurements of calcium clearance is probably not very great because their exact relation with tubular reabsorption has not been worked out. Comparison of data with Figure 6 may give some information on the renal or extrarenal origin of disturbances of plasma calcium. The ratio of calcium to creatinine clearance in healthy adults is in the order of 5 X 10-3.

Glomerular filtration rate and UCa V Equation (5) shows that a 50 per cent reduction of glomerular filtration rate has the same effect on plasma calcium as a doubling of extrarenal load (which equals UCaV). This demonstrates that the same variation in extrarenal load leads to a much greater variation in serum calcium when G.F.R. is low than- when G.F. R. is normal. That may be the reason that patients with sarcoidosis or myelomatosis who often have decreased G.F.R. tend to become hypercalcaemic on an increased extrarenal calcium load (Nordin and Peacock, 1969). There is, however, not much information about the renal tubular handling of calcium in chronic renal failure (Stanbury, 1968).

HOMEOSTASIS AND THE BONE Calcium ions can be taken up into or released from the bone in different ways (Figure I) (Marshall, 1969; Vaughan, 1970). They may be trapped in the bone for a long time by bone formation until released by osteoclastic bone resorption. They may be taken up by the bone for a very short time in the process of physiochemical exchange with calcium ions or other ions in crystals at the bone surface. This is short-term exchange (Marshall, Rowland and Jowsey, 1959). There may exist a continuous scale of processes of calcium replacement through the bone, from fast physiochemical exchange to slow structural remodelling, which may be characterised by the period of time during which, calcium ions stay buried in the bone. One of the intermediate types may be osteolysis (Belanger, 1965) .The sum of these exchange processes is the turnover of extracellular calcium through the bone (Heaney and Whedon, 1958; Marshall, 1969; Harris and Heaney, 1970). Quantitative approaches to the problem of bone turnover are heterogenous. There are morphometric methods (Jowsey et aI, 1965), kinetic studies or simple measurements of alkaline phosphatase or hydroxyproline.

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Kinetic studies are discussed elsewhere in this volume. The many models used to analyse the disappearance of calcium tracers from the blood traditionally aimed at deriving a value (mass/time) that should correspond with bone formation rate (Heaney and Whedon, 1958; Marshall, 1969; Harris and Heaney, 1970). There is no proof that these values measure bone formation rate and they probably do not. But it is questionable if the real information we are obtaining when using calcium tracers does indeed concern bone formation rate. It has been pointed out above that bone turnover rate is the sum of many turnover processes characterised by their turnover time. Marshall has shown that metabolic changes probably involve proportionate changes in all the rates of calcium transfer between the body and the blood, independent of their individual turnover time. He termed the sum of bone formation rate and of the more rapid processes of calcium transfer to bone 'addition rate', and pointed out that tracer data not only give quantitative information about addition rate but also about the relation between addition rate and turnover time. Marshall indicated how his hypothesis can be experimentally verified with kinetic studies. His approach (Marshall et ai, 1959; Marshall, 1964, 1969) probably allows to use results from kinetic studies not as unspecific estimates of bone formation rate but as quantitative measurements of internal calcium transfer, and its relation to turnover time. Combination of this information with information gained from histological studies may in the future give information about the relation between structural bone remodelling and the role of bone in the maintenance of calcium homeostasis. In a steady state, bone formation minus bone resorption equals calcium ingested minus calcium excreted (Harris and Heaney, 1970), and therefore kinetic data have been used to calculate bone resorption. The exact relation, however, between tracer data and bone formation is not known, and such data should be handled with care.

Alkaline phosphatase and bone remodelling Neither kinetic studies nor bone biopsies can easily be used in sequential studies or in screening of groups of patients. Measurements of alkaline phosphatase in plasma (Posen, 1967) and the rate of hydroxyproline excretion (Prockop and Kivirikko, 1967) in the urine are somewhat more appropriate for these purposes. Alkaline phosphatase is assayed by measuring the rate at which artificial substrates are split at an alkaline pH. The current laboratory methods differ in many details and the results are not interchangeable. The usefulness of the methods devised to differentiate between phosphatases of different origin in the body has still to be demonstrated (Newton, 1967; Posen, 1967). In the bone, alkaline phosphatase is associated with active calcification (Bourne, 1956) but the correlation between alkaline phosphatase in the plasma and bone turnover is poor. Yet alkaline phosphatase is higher in infants and children and during pubertal growth than in adults (Posen, 1967). Normal values have to be defined in relation to age and sex (Dent and Harper, 1962).

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A raised alkaline phosphatase is often found in diseases characterised by a local or general increase in bone turnover, such as Paget's disease, fractures, tumours, thyrotoxicosis, and sometimes in Cushing's disease. It also occurs in hyperparathyroidism and in rickets or osteomalacia. In these diseases the first sign of benefit from treatment may be an initial further rise of alkaline phosphatase (Fourman and Royer, 1968). A congenital disorder, hypophosphatasia is associated with a defect of mineralisation (Fraser, 1957). In contrast to hydroxyproline excretion, bone alkaline phosphatase activity in plasma has never been shown to change rapidly in hourly sequential studies. Hydroxyproline and bone remodelling Hydroxyproline is an amino acid present almost exclusively in the collagen. Bone matrix contains 57 per cent of total body collagen. Some of the hydroxyproline-containing soluble peptides synthesised during bone matrix formation are 'spilled over' in the body fluids and are excreted in the urine. Large amounts of hydroxyproline are released during collagen breakdown. Hydroxyproline is not re-utilised in the body. When the patient is on a collagen-poor diet, the urinary excretion of free and peptide-bound hydroxyproline reflects the combined rates of formation and breakdown of collagen (Prockop and Kivirikko, 1967). The retentate, high-weight polypeptide fraction of urine hydroxyproline obtained after dialysis of urine, may be more specifically related to collagen biosynthesis (Krane, Munoz and Harris, 1970). Hydroxyproline excretion may be raised with increased extraskeletal collagen turnover after burns, in psoriasis or in Cushing's disease. However, most of the variations in hydroxyproline excretion occurring in healthy persons are associated with variations of bone turnover (Prockop and Kivirikko, 1967), though the correlation is not close (Klein et aI, 1964). Hydroxyproline excretion varies with age, and in children there is a close association with rates of growth (Pappas et aI, 1971). If hydroxyproline assays are used as a diagnostic tool, age, sex, rate of growth and state of sexual maturity must be considered (Zorab, 1969). (Table 4). Serial measurements of urine hydroxyproline have been very useful in sequential monthly and even hourly assessment of the effect of various agents on bone metabolism Table 4. Normal Urinary Total Hydroxyproline Excretion (mgj24 hr) Boys Age

~

_ _ _ _..A--_ _ _ _- ,

(yr)

No.

Mean

s.d.

s.e.m,

11 12 13 14 15 16 17,18

52 51 48 44 46 34 35

68'6 78·0 93·2 110'0 106-4 81·1 55·1

32·5 29-4 43-9 48·0 41'6 32·0 24·0

4·5 4·1 6·3 7·2 6'1 5·5 4·1

Girls r------..A-------, Mean s.d. s.e.m. No. 38 53 52 60 48 53 67

54·7 69'1 51'1 38·0 31·1 31·7 27·1

27-6 30·2 25·8 17·7 12-9 13·2 14·5

4·5 4·1

3-6 2·3 1·9 1·8 1·8

Reproduced, with permission, from Zorab, P. A. (1969) Normal creatinine and hydroxyproline excretion in young persons. Lancet, ii, 1164-1165.

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and diseases (Bijvoet, van der Sluys Veer and Jansen, 1968; Bijvoet et ai, 1972). Studies in which alkaline phosphatase and hydroxyproline change in opposite directions (Smith, 1969) or in which the ratio of retentate fraction and total hydroxyproline excretion varies (Krane et ai, 1970), may be interesting. Calcium homeostasis and bone turnover Neither the level of plasma calcium nor the net differences between calcium ingested and calcium excreted-reflecting the bone-calcium balance-give information about the absolute rates of calcium uptake into and release from the skeleton. The body can counteract external challenges to the homeostasis of plasma calcium, modulating the relative rates of calcium uptake and release in bone (Talmage, 1956; Belanger, 1965; Phang et ai, 1969; Harris and Heaney, 1970). The sensitivity of bone to the modulating effects of hormones is probably not constant. Frost (1964, 1966) has pointed out that variation in bone resorption and formation rates are generally due to variation in the number of cells involved. One would therefore expect the net effect of a modulating hormonal influence to be greater the more cells participated in it. This interrelation between prevailing rate of bone remodelling and efficiency of bone-mediated control of calcium homeostasis can be illustrated. Is has, for instance, been shown that there is a positive relation between the effect of calcitonin on plasma calcium and bone remodelling rate (Bijvoet et al, 1968). It has been observed that myxoedaemic children with low bone remodelling rates cannot cope adequately with artificial hyper- or hypo-calcaemia. Their plasma calcium levels depend to a large extent on their intake of calcium (Lowe, Bird and Thomas, 1962; Klotz and Kanovitz, 1966).

HOMEOSTASIS AND THE GUT Calcium absorption In the United States and the United Kingdom the calcium content of diet varies from 600 to 1000 mg per day. Calcium intake with drinking wateroften neglected-may range from nil to 200 mg per day. In normal adults less than half of the dietary calcium is absorbed (Krane, 1970). The efficiency of absorption partly depends upon the actual intake, but also on the previous dietary history, on the state of calcium in the food, and on phosphate uptake (Nicolaysen, 1943; Maim, 1958; Avioli, McDonald and Lee, 1965). Calcium absorption requires vitamin D. It is increased in sarcoidosis and with excess of parathyroid hormone or growth hormone. In a number of patients with so called idiopathic hypercalciuria, hyperabsorption occurs. Absorption is subnormal in vitamin D deficiency, in renal failure, and sometimes in patients with glucocorticoid excess (Fourman and Royer, 1968). Faecal calcium is not simply the unabsorbed fraction of total calcium intake, because calcium is secreted into the gut. Measurements with intravenously administered isotopes of calcium give values in the order of 200 mg per day (Harris and Heaney, 1970).

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There are two types of measurement of calci urn absorption: 1. A simple oral dose of a radioactive isotope of calcium is given with a variable amount of carrier. The specific activity of plasma, or urinary isotope excretion, or the radio-activity of the whole body or parts of it, are then measured (Avioli, McDonald and Lee, 1965). There are many variations of this method; they give only approximate answers and are not standardised, and will not be discussed here. 2. In calcium balance studies, calcium absorption is calculated from the difference between dietary and faecal calcium. Allowance may be made for calcium secretion into the gut by measuring the faecal excretion of intravenously administered radioactive isotopes of calcium (Albright and Reifenstein, 1948; Hargreaves and Rose, 1965; lsaksson, Lindholm and Sjogren, 1967; Isaksson and Sjogren, 1967).

Metabolic balance technique Balance studies have two aims: first, to measure the net difference between total rate of calcium ingestion and total rate of calcium output; and secondly, to define separately the rates of all single processes of calcium transfer charted in Figure I. The balance study should then be accompanied by kinetic studies. The word 'balance' should not refer to weighing but to book-keeping. All results are arrived at by calculating differences and this req uires very accurate measurements. Therefore, calcium intake and faecal excretion, which are discontinuous processes, can only be expressed as continuous rates when assessed over long periods (Isaksson et ai, 1967; Isaksson and Ohlsson, 1967; Isaksson and Sjorgren, 1967). Three to six-day collection periods seem acceptable provided food markers (carmine red) are used to correct for time-lag between intake and faecal excretion (Rose, 1964). Accuracy can be increased by referring excretion rates to the excretion rate of an unabsorbable substance that is administered 'continuously' orally, such as CrZ03 (Whithy and Lang, 1960; Hargreaves and Rose, 1965). Dermal losses of calcium are not often measured (Whithy and Lang, 1965, Rose, 1964) but they may be important (lsaksson and Ohlsson, 1967). Constant composition of diet necessitates that all food should be prepared in advance, divided into eq ual rations and deepfrozen until used. Only distilled water should be used for drinking and food preparations. Random dietary samples have to be analysed because calculated and actual composition of the diet differ. As dietary habits influence the efficiency of calcium absorption, the diet should conform to the habitual diet obtained from a history (Nicolaysen, 1943; MaIm, 1958). It is evident that the patient should be in a steady state; therefore no change should be made in his circumstances and activities, and he should not be convalescing from a disease or fracture. His collaboration and well-informed interest is vital.

Bone balance and calcium balance When the idea is accepted that an important function of the skeleton is to serve as a store and buffer for extracellular homeostasis, it will be clear that net gains and losses of calcium in the skeleton never exactly match. Short periods of negative or positive balance will alternate.

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One should distinguish between long term net gain or loss of bone calci urn related to structural changes, growth, ageing and metabolic changes in th e body on the one hand, and short-term rapid changes in the equilibrium between gain and loss in the maintenance of homeostasis on the other hand. The latter are superimposed upon the long-term effects, and hence a positi ve or negative balance observed in short studies may be transient and cannot be multiplied by an appropriate factor to obtain the net gain and loss of bone over years. It is also dangerous to extrapolate effects of drugs on calcium balance observed to persist even over several weeks. Bone resorption and formation may be mutually linked phases of a cyclic bone remodelling system. Once a bone remodelling system is disturbed, a transient state may set in which may require several months to regain a new steady state at a different or at the original level (Frost, 1966, 1969). For these reasons, metabolic balance studies should not aim at deriving information about bone balance but at trying to define separately the rates of the various processes of calcium transfer in the body. They should preferably be used to study sequential changes brought about in these rates, by welldefined factors. Net long-term gain or loss of bone or calcium over months or years can only be measured by direct measurements of bone mass. Various promising approaches have been made, such as morphometry (Jowsey et al 1965; Frost, 1969), assessing metacarpal thickness (Meema and Meema, 1963; Morgan et al, 1967) and densitometry by direct measurement of photon absorption in the bone (Cameron and Sorenson, 1963).

CONCLUSION We have tried to define some provisional concepts used in the interpretation of laboratory data. Good data should not illustrate but improve such concepts (Bernard, 1865).

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