Upsala J Med Sci 92: 147-176, 1987 Optimal Discrimination of Mild Hyperparathyroidism with Total Serum Calcium, Ionized Calcium and Parathyroid Hormone Measurements Lars Benson', Sverker Ljunghall', Torgny Groth3, Hans Falk3, Andreas Hvarfnef, Jonas Rastad4, Leif Wide' and Goran Akerstrom4 Departmenls of 'Clinical Chemistry, 2Medicine, 4Surgery, University Hospital, and 'Unit of Biomedical Systems Analysis, Uppsala University, Uppsala, Sweden A B S T R A C T T h e s e r u m c o n c e n t r a t i o n s o f calcium, a l b u m i n a n d p a r a t h y r o i d hormone ( P T H ) a n d t h e plasma levels o f i o n i z e d calcium w e r e d e t e r m i n e d in 1 2 4 h e a l t h y s u b j e c t s , 8 9 p a t i e n t s w i t h p r i m a r y h y p e r p a r a t h y r o i d i s m ( H P T ) , 2 3 o f whom h a d t h e s y n d r o m e o f m u l t i p l e e n d o c r i n e neoplasia t y p e 1 (MEN-1) a n d 4 3 p a t i e n t s who h a d hypercalcaemia o f o t h e r causes t h a n H P T ( n o n - H P T ) , in most cases d u e t o w i d e s p r e a d malignancies. T h e t o t a l s e r u m calcium was c o r r e c t e d f o r t h e s e r u m a l b u m i n c o n c e n t r a t i o n (CaM). H e a l t h y females o v e r t h e age o f 5 0 h a d h i g h e r CaM, t h a n y o u n g e r females a n d t h e women o f a l l ages also h a d , h i g h e r s e r u m P T H l e v e l s t h a n males. F o r a l l s t u d y g r o u p s b o t h t h e i n t r a - a n d i n t e r - d i u r n a l v a r i a t i o n s w e r e small f o r a l l t h e s t u d i e d v a r i a b l e s . D i s c r i m i n a n t f u n c t i o n a n d optimal d i s c r i m i n a t o r y l i m i t s w e r e c a l c u l a t e d w i t h t h e h e l p o f c o m p u t e r p r o g r a m s . A c o n s i d e r a t i o n o f a l l t h e i n d i v i d u a l s in t h e d i s c r i m i n a n t a n a l y s i s , r e v e a l e d t h a t measurements o f CaM alone s e p a r a t e d most H P T p a t i e n t s b o t h f r o m t h e h e a l t h y s u b j e c t s a n d f r o m t h e non-HPT p a t i e n t s . However, w h e n o n l y those who h a d b o r d e r l i n e values ( d e f i n e d as CaM between 2 . 4 5 a n d 2 . 7 5 m m o l / l ) w e r e i n c l u d e d it t u r n e d o u t t h a t measurements o f i o n i z e d calcium m a r k e d l y i m p r o v e d t h e d e l i n e a t i o n o f m i l d H P T f r o m t h e h e a l t h y s u b j e c t s a n d t h a t , in a d d i t i o n , PTH measurements h e l p e d t o e x c l u d e those w i t h n o n - H P T hypercalcaemia. T h e optimal d i s c r i m i n a t o r y l e v e l s o f s e r u m calcium w e r e c a l c u l a t e d as t h e l e v e l s w h i c h c a u s e d t h e minimum loss in t e r m s o f m i s c l a s s i f i c a t i o n w h e n a t t e n t i o n was p a i d t o t h e r e l a t i v e i m p o r t a n c e o f false p o s i t i v e t o false n e g a t i v e c l a s s i f i c a t i o n s a n d t o t h e p r e v a l e n c e o f HPT. T h e optimal d i s c r i - m i n a t o r y l e v e l f o r serum calcium f o r a w e i g h t i n g r a t i o between false p o s i t i v e t o false n e g a t i v e of 1:1, a n d a p r e v a l e n c e of HPT o f 1 &, was c a l c u l a t e d t o b e 2 . 6 8 mmol/I a n d f o r a p r e v a l e n c e o f 5 0 % 2 . 5 6 mmol/I. In t h e l a t t e r s i t u a t i o n a w e i g h t i n g r a t i o o f 1O:l f o r false p o s i t i v e t o false n e g a t i v e g a v e a level o f 2 . 6 3 m m o l l l w h i l e a w e i g h t i n g r a t i o o f 1 : 1 0 c o r r e s p o n d e d t o a n optimal d i s c r i m i n a t o r y l e v e l o f 2 . 4 7 mmol/I. 10-878572 147 I N T R O D U C T I O N P r i m a r y h y p e r p a r a t h y r o i d i s m ( H P T ) i s a common d i s o r d e r , k n o w n t o a f f e c t o n e p e r c e n t o f t h e p o p u l a t i o n a b o v e 60 y e a r s o f age a n d o c c u r r i n g a t e v e n h i g h e r f r e q u e n c i e s in o l d e r i n d i v i d u a l s (11,36,49). T h e high p r e - valence o f t h e disease c a l l s for a c c u r a t e s c r e e n i n g a n d d i a g n o s t i c methods w h i c h c a n e a s i l y p r o v i d e a n i d e n t i f i c a t i o n o f t h e diseased p a t i e n t s w i t h a minimal n u m b e r o f misclassifications. T h e i d e n t i f i c a t i o n o f p a t i e n t s w i t h H P T in t h e c l i n i c a l r o u t i n e r e l i e s p r i m a r i l y o n t h e d e m o n s t r a t i o n o f hypercalcemia. In a p r e v i o u s s t u d y s i n g l e measurements o f t o t a l s e r u m calcium w e r e u s e d t o c a l c u l a t e optimal d i s c r i - m i n a t i n g l i m i t s f o r t h e d i a g n o s i s o f p r i m a r y HPT ( 1 8 ) . In t h e p r e s e n t r e p o r t we h a v e e x t e n d e d t h i s a n a l y s i s t o i n c l u d e measurements o f t h e s e r u m con- c e n t r a t i o n s o f p a r a t h y r o i d hormone ( P T H ) a n d of ionized calcium, d e t e r m i n a - t i o n s o f w h i c h h a v e become more w i d e l y a v a i l a b l e during r e c e n t y e a r s . P r a c t i c a l l y a l l s t u d i e s d e a l i n g w i t h t h e d i a g n o s i s o f H P T a r e based o n m a t e r i a l s w h e r e t h e m a j o r i t y o f p a t i e n t s h a v e m a r k e d hypercalcaemia. In c l i n i c a l p r a c t i c e , h o w e v e r , most p a t i e n t s w i t h s u s p e c t e d H P T nowadays h a v e o n l y m i l d l y e l e v a t e d s e r u m calcium v a l u e s ( 2 0 . 5 0 ) . T h e p r e s e n t s t u d y t h e r e f o r e d e v o t e s p a r t i c u l a r a t t e n t i o n t o t h e p o t e n t i a l v a l u e o f i o n i z e d calcium a n d PTH measurements in t h e d e l i n e a t i o n o f m i l d HPT. In many i n s t a n c e s t h e r e may n o t b e a g r e a t need t o e s t a b l i s h t h e d i a g n o s i s o f HPT in s u c h b o r d e r l i n e cases. Sometimes, h o w e v e r , a p r e c i s e c l a s s i f i c a t i o n c o u l d b e i m p o r t a n t . For example we r e c e n t l y r e p o r t e d ( 2 ) t h a t in t h e d o m i n a n t l y i n h e r i t e d s y n d r o m e o f m u l t i p l e endocrine, neoplasia t y p e 1 (MEN-1) H P T i s a p p a r e n t l y t h e f i r s t m a n i f e s t a t i o n . T h e d e m o n s t r a t i o n o f m i l d H P T t h e r e f o r e i s t h e e a r l i e s t o p p o r t u n i t y t o disclose t h e c a r r i e r o f t h e MEN-1 t r a i t . P a t i e n t s w i t h r e c u r r e n t r e n a l stones c o n s t i t u t e a n o t h e r g r o u p w h e r e it i s highly d e s i r a b l e t o o b t a i n d e f i n i t e e v i d e n c e f o r o r a g a i n s t HPT d e s p i t e u n d e c i d e d hypercalcemia . A n o t h e r c l i n i c a l p r o b l e m in t h e d i a g n o s i s o f HPT c o n s i s t s o f t h e e x c l u - s i o n o f o t h e r causes o f hypercalcemia, p r i m a r i l y m a l i g n a n t d i s o r d e r s . A l t h o u g h these a r e g e n e r a l l y e v i d e n t c l i n i c a l l y , a d d i t i o n a l i n v e s t i g a t i o n s a r e sometimes r e q u i r e d . I48 REFERENCE SAMPLE GROUPS H e a l t h y s u b j e c t s From a h e a l t h s u r v e y in Uppsala c o u n t y 98 a p p a r e n t l y h e a l t h y indivi- d u a l s w e r e r e c r u i t e d t o r e p r e s e n t a " h e a l t h y r e f e r e n c e sample g r o u p " ; 5 2 men a n d 46 women, a g e d between 16 t o 9 2 , w i t h a mean age of 46 f. 20 y e a r s (mean f S D ) , a n d equal d i s t r i b u t i o n o f t h e sexes in a l l age g r o u p s . T w e n t y - s i x a p p a r e n t l y h e a l t h y employees from t h e h o s p i t a l s t a f f , 10 men a n d 1 6 women, a g e d between 20-60 y e a r s , p a r t i c i p a t e d in s t u d i e s o f t h e i n t r a - a n d , i n t e r - d i u r n a l v a r i a t i o n s . Patients w i t h H P T : 89 c o n s e c u t i v e p a t i e n t s o p e r a t e d f o r HPT, 2 7 men a n d 62 women, a g e d between 19-83 w i t h a mean o f 60 f 17 y e a r s w e r e s t u d i e d b e f o r e o p e r a t i o n : 66 o f them h a d s p o r a d i c HPT a n d s e r u m calcium was normalized p o s t o p e r a t i v e l y in a l l cases. T h e o t h e r 2 3 , 1 6 women a n d seven men, h a d H P T as a p a r t o f MEN-1, n i n e o f them h a d p e r s i s t e n t o r r e c u r r - e n t hypercalcaemia a f t e r p r e v i o u s o p e r a t i o n s . F i v e o f them also h a d a n e n d o c r i n e t u m o r o f t h e pancreas, in t h r e e cases w i t h l i v e r metastases. Neck e x p l o r a t i o n c o n f i r m e d p a r a t h y r o i d h y p e r p l a s i a . S u b t o t a l o r t o t a l p a r a t h y - roidectomy w i t h a u t o t r a n s p l a n t a t i o n was p e r f o r m e d . Serum calcium r e t u r n e d t o t h e normal r a n g e p o s t o p e r a t i v e l y in a l l cases. P a t i e n t s w i t h hypercalcemia o f o t h e r o r i g i n t h a n HPT ( N o n - H P T ] : F o r t y - t h r e e p a t i e n t s , 21 men a n d 2 2 women, aged 38-78 w i t h a mean age o f 60 f. 15 y e a r s h a d hypercalcemia o f c l i n i c a l l y o b v i o u s causes o t h e r t h a n HPT. M a l i g n a n c y was t h e most common cause, b e i n g e n c o u n t e r e d in 3 0 pa- t i e n t s , 13 men a n d 17 women. Renal c a n c e r was seen in s e v e n cases, f i v e h a d cancer mammae, f i v e h a d lung c a n c e r , f i v e h a d myeloma, t h r e e cancer o f t h e pancreas, t h r e e lymphomas, one h a d t h y r o i d c a n c e r a n d one p a t i e n t h a d a leiomyosarcoma. In most cases b o n e metastases w e r e e v i d e n t . O t h e r causes of hypercalcemia w e r e e n c o u n t e r e d in 13 p a t i e n t s , s i x men a n d s e v e n women. Sarcoidosis was seen in s e v e n p a t i e n t s , whose s e r u m calcium v a l u e s n o r m a l i z e d u p o n t r e a t m e n t w i t h s t e r o i d s . T w o p a t i e n t s h a d t h y r o t o x i - cosis, a n d became normocalcemic in response t o medical t r e a t m e n t . Immobili- zation d u e t o t e t r a p l e g i a was t h e cause o f hypercalcaemia in f o u r p a t i e n t s . 149 L A B 0 R A T 0 RY MET H OD S Blood specimens w e r e c o l l e c t e d b e t w e e n 07.00-09.00 o n t h e m o r n i n g f o l l o w i n g a n o v e r n i g h t f a s t . F o r each p a t i e n t t h e mean v a l u e o f a l l s u c h measurements was c a l c u l a t e d a n d u s e d as t h e basal value. F o r t h e s t u d y o f v a r i a t i o n during t h e d a y , specimens w e r e also c o l l e c t e d b e f o r e meals a t 1 2 . 0 0 a n d 16.00 h o u r s . No d i e t r e s t r i c t i o n s a p a r t f r o m a b a n o n m i l k a n d cheese w e r e imposed. I o n i z e d calcium ( C a l l : Whole b l o o d was c o l l e c t e d a n a e r o b i c a l l y in 5 ml h e p a r i n i z e d t u b e s a n d a n a l y z e d w i t h i n a few h o u r s f o r i o n i z e d calcium w i t h a n ion-selective e l e c t r o d e ( M i c r o l y t e , Kone I n s t r u m e n t s , F i n l a n d ) . All samples w e r e measured in d u p l i c a t e . T h e a n a l y z e r has a n automatic t h r e e p o i n t c a l i b r a t i o n p r o c e d u r e u s i n g w a t e r s t a n d a r d s a d j u s t e d f o r i o n i c s t r e n g t h a n d p H . A f t e r each sample a m i d d l e s t a n d a r d i s measured f o r assessment o f drift. T h e t e m p e r a t u r e o f t h e e l e c t r o d e b l o c k i s m a i n t a i n e d a t 3OoC. In p a t i e n t samples t h e a v e r a g e a n a l y t i c a l w i t h i n - r u n s t a n d a r d d e v i a - t i o n was 0 . 0 1 2 mmol/I a t t h e level o f 1 . 1 5 mmol/I a n d 0.019 mmol/l a t t h e l e v e l o f 1.45 mmol/l. T h e l i f e - s p a n o f a n e l e c t r o d e i s 3 - 6 m o n t h s . A f t e r c h a n g e o f e l e c t r o d e , t h e v a l u e s (n=178) f r o m a r e f e r e n c e p o p u l a t i o n of h e a l t h y i n d i v i d u a l s w e r e compared w i t h p r e v i o u s v a l u e s (n=100) in t h e same i n d i v i d u a l s . A d i f f e r e n c e o f 0.01 mmol/l was o b s e r v e d f o r t h e mean v a l u e s , w i t h o u t c h a n g e in i m p r e c i s i o n . T h e r e a r e n o e x t e r n a l c o n t r o l s t a n d a r d s f o r i o n i z e d calcium, a n d e x t e r n a l q u a l i t y assessment schemes h a v e s t r e s s e d t h e u s e o f p r o t e i n - e n r i c h e d s o l u t i o n s ( 4 4 ) . In t h e absence o f a n e x t e r n a l c o n t r o l sample, r e c o n s t i t u t e d l y o p h i l i z e d c o n t r o l sera ( V a l i d a t e , General D i a g n o s t i c s ) f r o m t h e same b a t c h - a c t u a l l y n o t m a n u f a c t u r e d f o r t h i s p u r p o s e a n d n o t always w i t h i n t h e r e f e r e n c e r a n g e f o r h e a l t h y s u b j e c t s , - h a v e b e e n u s e d as a n e x t e r n a l c o n t r o l o v e r time w i t h a C V o f 1 . 4 % a t 1 . 2 0 mmol/I a n d 2 . 3 % a t 1 . 6 0 mmol/l. No e f f o r t was t a k e n t o i m p r o v e s t a n d a r d i z a t i o n o f w a t e r , t e m p e r a t u r e , p H o r g a s c o n t e n t ( 9 ) . Serum was o b t a i n e d a f t e r c l o t t i n g a n d c e n t r i f u g a t i o n , a n d was e i t h e r a n a l y z e d in t h e o r d i n a r y l a b o r a t o r y r o u t i n e t h e same d a y , o r s t o r e d a t +4O o v e r n i g h t a n d a n a l y z e d t h e n e x t d a y . T o t a l s e r u m calcium ( C a T ) c o n c e n t r a t i o n s w e r e d e t e r m i n e d by a n atomic a b s o r p t i o n t e c h n i q u e ( Perkin-Elmer 3 0 3 0 ) . T h e a n a l y t i c a l p r o c e d u r e f o r d e t e r m i n a t i o n o f s e r u m calcium h a d a n a v e r a g e w i t h i n - r u n s t a n d a r d d e v i a - t i o n o f 0 . 0 3 8 mmol/l, a n d a n a v e r a g e b e t w e e n - r u n s t a n d a r d d e v i a t i o n o f 150 0.022 mmol/l, giving a t o t a l a n a l y t i c a l s t a n d a r d d e v i a t i o n o f 0.044 mmol/l. A l l v a l u e s r e f e r t o a c o n c e n t r a t i o n l e v e l o f 2.46 mmol/I, a n d t h e a n a l y t i c a l e r r o r was assumed t o b e t h e same f o r a l l c o n c e n t r a t i o n l e v e l s e x p r e s s e d as a c o e f f i c i e n t o f v a r i a t i o n (CV=0.018). T h e a n a l y t i c a l b i a s o f t h e p r o c e d u r e was a b o u t -1% compared w i t h t h e r e f e r e n d u m v a l u e o f t h e r e g i o n a l e x t e r n a l q u a l i t y assessment p r o g r a m . Serum a l b u m i n was d e t e r m i n e d by a b r o m c r e s o l - b i n d i n g t e c h n i q u e a n d c a l i b r a t e d w i t h p u r i f i e d human a l b u m i n s o l u t i o n . T h e a n a l y t i c a l w i t h i n - r u n s t a n d a r d d e v i a t i o n was 0.42 g / l a n d t h e b e t w e e n - r u n s t a n d a r d d e v i a t i o n 0.71 g / l , giving a t o t a l a n a l y t i c a l s t a n d a r d d e v i a t i o n o f 0.83 g / l . C o r r e c t i o n o f CaT f o r s e r u m a l b u m i n c o n c e n t r a t i o n ( C a M ) : A s s e r u m calcium i s b o u n d t o a l b u m i n , a c o r r e c t i o n ( m o d i f i c a t i o n ) o f s e r u m calcium v a l u e s was made f o r d e v i a t i o n s o f t h e a c t u a l a l b u m i n f r o m t h e r e f e r e n c e mean v a l u e o f 42 g / l by t h e f o l l o w i n g formula u s e d in o u r l a b o r a t o r y : CaM = CaT - 0.019 (S-albumin - 421 mmol/I Radioimmunoassay o f p a r a t h y r o i d hormone ( P T H ) in s e r u m Serum specimens w e r e k e p t a t - 2OoC until analyzed. T h e P T H concen- t r a t i o n was d e t e r m i n e d by a radioimmunoassay s y s t e m e m p l o y i n g 121 I- labelled b o v i n e P T H ( I n o l e x ) a n d sheep a n t i s e r u m ( S 478) a g a i n s t p o r c i n e a n d b o v i n e PTH. T h i s a n t i s e r u m r e a c t s w i t h a m i d - p o r t i o n (44-68) o f human P T H but has also a high a f f i n i t y ( 0 . 6 x 1013 I/mmol) f o r i n t a c t human PTH (21 1 . T h e assay p r o c e d u r e u s e d s o l i d phase-coupled a n t i - s h e e p - l g G t o s e p a r a t e b o u n d a n d f r e e labelled PTH. T h e s e r u m specimens a n d t h e a n t i s e r u m w e r e f i r s t i n c u b a t e d f o r 2 4 h a t 4OC followed by a 48 h i n c u b a t i o n w i t h l a b e l l e d PTH . Microsepharose-coupled h o r s e a n t i - s h e e p - l g G ( d e c a n t i n g suspension 2, Pharmacia AB, Sweden) was t h e n a d d e d a n d t h e i n c u b a t i o n was p r o l o n g - e d f o r 3 h. T h e p a r t i c l e s w e r e c e n t r i f u g e d d o w n during 5 m i n a t 2000 g a n d t h e p e l l e t was washed once w i t h saline c o n t a i n i n g 0.5 % Tween-20. B o v i n e PTH d i l u t e d in human sera w i t h low PTH l e v e l s was u s e d as labora- t o r y s t a n d a r d a n d t h e c o n c e n t r a t i o n o f P T H in human s e r u m was e x p r e s s e d in a r b i t r a r y u n i t s ( a r b U l l ) . A b o u t 2.5 a r b U w e r e e q u i v a l e n t t o 1 0 U N I B S C r e s e a r c h s t a n d a r d f o r human P T H 75/479. A l l specimens w e r e assay- e d in d u p l i c a t e . T h e t o t a l assay c o e f f i c i e n t o f v a r i a t i o n in 42 assays was 9.2%. w i t h a n a v e r a g e w i t h i n - a s s a y c o e f f i c i e n t of v a r i a t i o n o f 3.6% a t a l e v e l o f 0.7-0.8 a r b U/I. 151 T h e disappearance o f s e r u m P T H f o l l o w i n g p a r a t h y r o i d e c t o r n y was i n v e s - t i g a t e d in 1 0 p a t i e n t s w i t h adenomatous H P T , basal values o f 0 . 8 1 - 1.24 a r b U / l a n d a normal k i d n e y f u n c t i o n . A mean r e d u c t i o n o f 17% was f o u n d w i t h i n t h e f i r s t 1 5 m i n u t e s , w h i c h d e m o n s t r a t e d a c a p a b i l i t y o f t h e assay system r a p i d l y t o d e t e c t changes in s e r u m c o n c e n t r a t i o n s ( F i g . 1 ) . 0 -5 -10 -15 - 2 0 -25 4 Fig. 1 . R e d u c t i o n o f s e r u m p a r a t h y r o i d hormone ( P T H ) c o n c e n t r a t i o n s a f t e r p a r a t h y r o i d e c t o m y in 10 p a t i e n t s w i t h adenornatous h y p e r - p a r a t h y r o i d i s m . ( B a r s i n d i c a t e SEM). 152 C O M P U T A T I O N A L METHODS A l l s t a t i s t i c a l c a l c u l a t i o n s w e r e p e r f o r m e d o n a B A S F 7 / 7 3 - I B M / M V S computer system a t Uppsala U n i v e r s i t y Data C e n t e r . T h e S t a t i s t i c a l A n a l y s i s System p a c k a g e (SAS I n s t i t u t e I n c . , N o r t h C a r o l i n a U S A ) was u s e d f o r d e s c r i p t i v e s t a t i s t i c s (means a n d s t a n d a r d d e v i a t i o n s o f v a r i o u s r e f e r e n c e sample g r o u p s ; biological i n t r a - a n d i n t e r - d i u r n a l v a r i a t i o n ; c r o s s - p l o t t i n g ; s t a t i s t i c a l g o o d n e s s - o f - f i t - t e s t s ) , l i n e a r r e g r e s s i o n analysis; a n d n o n - l i n e a r parameter fitting. Stepwise d i s c r i m i n a n t a n a l y s i s was p e r f o r m e d w i t h t h e BMDP p r o g r a m package (Biomedical Computer Programs, P-series, U n i v e r s i t y o f C a l i f o r n i a , 1 9 7 7 ) . A p r o g r a m f o r a n a l y s i s o f v a r i a n c e o n a LUXOR ABC-80 c o m p u t e r was u s e d t o c a l c u l a t e t h e a n a l y t i c a l w i t h i n - , a n d b e t w e e n - r u n v a r i a t i o n . Optimal d i s c r i m i n a t o r y l i m i t s w e r e c a l c u l a t e d w i t h t h e h e l p o f a p r o g r a m developed a t t h e U n i t o f Biomedical Systems A n a l y s i s ( 1 8 ) . T h i s p r o g r a m ca Icu lates : ( a ) t h e e x p e c t e d f r e q u e n c y o f false n e g a t i v e a n d false p o s i t i v e outcomes in c o n n e c t i o n w i t h c l a s s i f i c a t i o n , u s i n g a s p e c i f i e d d i s c r i m i n a t o r y level; ( b ) a measure o f loss r e l a t e d t o misclassification, ( c ) t h e optimal d i s c r i m i n a t o r y l i m i t , ( e ) , a n d ( d ) t h e d i a g n o s t i c s e n s i t i v i t y , s p e c i f i c i t y a n d t h e p r e d i c t e d v a l u e o f a p o s i t i v e a n d n e g a t i v e t e s t r e s u l t . Input data t o t h e p r o g r a m a r e : ( a ) f r e q u e n c y d i s t r i b u t i o n s o f t h e r e f e r e n c e p o p u l a t i o n s ; in t h i s case d i s t r i b u t i o n s r e p r e s e n t i n g h e a l t h y i n d i v i d u a l s , p a t i e n t s w i t h s p o r a d i c HPT o r H P T as p a r t o f MEN-1, as well as p a t i e n t s w i t h non-HPT hypercalcemia ; ( b ) p r e v a l e n c e o f t h e diseases, e x p r e s s e d as n u m b e r - r a t i o s ; ( c ) n u m e r i c a l w e i g h t s , W1 a n d W2, r e p r e s e n t i n g t h e r e l a t i v e c o s t s o f m a k i n g misclassifications; [ d ) p r e - a n a l y t i c a l a n d a n a l y t i c a l v a r i a t i o n e x p r e s s e d as c o e f f i c i e n t s o f v a r i a t i o n : “pre-a a n d C V a , r e s p e c t i v e l y ; T h e v a r i a n c e s o f f r e q u e n c y d i s t r i b u t i o n s r e p r e s e n t i n g t h e d i f f e r e n t r e f e r - ence p o p u l a t i o n s a r e calculated f r o m : 153 2 2 2 + ' a t o t a l - b i o l + p r e - a - 2 a n d t h e r e f o r e 2 - 2 - 2 b i o l - t o t a l (' p r e - a w h e r e S i s t h e t o t a l b i o l o g i c a l s t a n d a r d d e v i a t i o n ( i n c l u d i n g i n t r a - , a n d i n t e r - i n d i v i d u a l v a r i a t i o n ) ; Spre-a i s t h e p r e - a n a l y t i c a l s t a n d a r d d e v i a t i o n , i.e. t h e v a r i a t i o n r e l a t e d t o specimen h a n d l i n g ; a n d Sa i s t h e a n a l y t i c a l s t a n d a r d d e v i a t i o n . In t h e p r o g r a m t h e c a l c u l a t i o n s a r e p e r f o r m e d t o estimate t h e "tail-areas" c u t - o f f by a s p e c i f i e d d i s c r i m i n a t o r y l i m i t f r o m t h e d i f f e r e n t d i s t r i b u t i o n s , giving v a l u e s f o r t h e n u m b e r o f false p o s i t i v e s ( F P ) a n d false n e g a t i v e s ( F N ) . F N ) / $ O w h e r e 4 i s t h e loss u n d e r ideal c o n d i t i o n s , b i o l A s p r e v i o u s l y d e s c r i b e d ( 1 8 ) t h e loss i s c a l c u l a t e d as A = ( W l x F P + W 2 x CVa=O a n d CVpre-a =O . Since t h e w e i g h t i n g f a c t o r s a r e g i v e n in r e l a t i v e n u m b e r s , t h e loss s h o u l d b e r e g a r d e d as a r e l a t i v e loss. T h e c a l c u l a t i o n s a r e automatically r e p e a t e d f o r a n u m b e r o f d i f f e r e n t values f o r t h e d i s c r i m i n a t o r y level, in o r d e r t o allow d e t e r m i n a t i o n o f t h e optimal value. RESULTS D e s c r i p t i v e s t a t i s t i c s : T a b l e 1 summarizes t h e mean v a l u e s f o r t h e s t u d i e d v a r i a b l e s in a l l t h e f i v e g r o u p s o f s u b j e c t s . T h e mean calcium v a l u e s w e r e h i g h e r in t h e p a t i - e n t s w i t h n o n - H P T hypercalcaemia t h a n in t h e H P T p a t i e n t s . T h e HPT p a t i e n t s h a d a h i g h e r mean v a l u e f o r P T H t h a n a l l o t h e r g r o u p s . T h e r e w e r e small, but s t a t i s t i c a l l y s i g n i f i c a n t , age- a n d s e x - r e l a t e d d i f f e r e n c e s w i t h i n t h e g r o u p o f normal s u b j e c t s ( T a b l e 2 ) . Women o v e r t h e age o f 5 0 h a d h i g h e r CaM values t h a n y o u n g e r females. O n t h e o t h e r h a n d males o v e r t h e age o f 5 0 showed lower CaT ( b u t n o t CaM) v a l u e s t h a n did t h e y o u n g e r men. No d i f f e r e n c e s w e r e n o t e d f o r C a l b e t w e e n t h e s u b - g r o u p s . Females o f a l l ages h a d c l e a r l y h i g h e r v a l u e s f o r P T H t h a n males. T h e a v e r a g e t o t a l i n t r a - d i u r n a l v a r i a t i o n s ( i n c l u d i n g b i o l o g i c a l , p r e - a n a l y t i c a l a n d a n a l y t i c a l ) f o r t h e d i f f e r e n t g r o u p s a r e g i v e n in T a b l e 3. T h e r e w e r e n o s i g n i f i c a n t v a r i a t i o n s o v e r t h e d a y in a n y o f these g r o u p s f o r a n y o f t h e v a r i a b l e s s t u d i e d . T a b l e 4 g i v e s t h e i n t r a - i n d i v i d u a l v a r i a t i o n s b o t h w i t h i n a n d b e t w e e n d a y s . A s c a n b e seen in t h e T a b l e t h e v a r i a t i o n s f o r t h e calcium measure- 154 ments w e r e somewhat g r e a t e r in t h e hypercalcaemic i n d i v i d u a l s t h a n in t h e h e a l t h y s u b j e c t s . N a t u r a l l y , f o r a l l s t u d i e d v a r i a b l e s t h e i n t e r - d i u r n a l v a r i a t i o n s w e r e g r e a t e r t h a n t h e i n t r a - d i u r n a l but g e n e r a l l y t h e d i f f e r e n c e s were small. O n t h e b a s i s o f these measurements it c o u l d b e c a l c u l a t e d t h a t t h e biological SD f o r t h e C a l v a l u e s was 0.045 mmol/I f o r b o t h h e a l t h y s u b j e c t s a n d p a t i e n t s w i t h H P T w h i l e f o r CaM it was 0.055 m m o l / l in b o t h g r o u p s . S i m i l a r l y t h e b i o l o g i c a l v a r i a t i o n f o r P T H was c a l c u l a t e d t o b e 0.14 a r b U / I in t h e h e a l t h y s u b j e c t s a n d 0.09 a r b U / I in t h e p a t i e n t s w i t h H P T . T a b l e 1. Mean v a l u e , s t a n d a r d d e v i a t i o n ( S D ) , a n d s t a n d a r d e r r o r o f t h e mean (SEM) f o r t h e measured v a r i a b l e s in t h e d i f f e r e n t s u b j e c t g r o u p s . n Mean SD S EM Plasma i o n i z e d calcium ( m m o l / l ) ( C a t ) H e a l t h y 93 1.203 0.047 0.005 HPT, S p o r a d i c 52 1.400 0.17 0.024 H P T /MEN 12 1.416 0.133 0.038 Non-HPT 43 1.513 0.242 0.037 T o t a l s e r u m calcium ( m m o l / l ] ( C a T ) H e a l t h y 98 2.425 0.082 0.009 H P T , S p o r a d i c 65 2.836 0.287 0.035 H P T / MEN 23 2.745 0.192 0.040 Non-HPT 43 3.103 0.538 0.081 Serum a l b u m i n l a / l l H e a l t h y 98 43.06 2.86 0.30 HPT, S p o r a d i c 65 38.50 3.83 0.28 H P T / M E N 23 39.78 4.55 0.62 Non-HPT 43 35.32 5.49 0.89 A l b u m i n - m o d i f i e d s e r u m calcium ( m m o l / l ) (CaM) Heal thy 98 2.415 0.079 0.008 HPT, S p o r a d i c 65 2.908 0.301 0.037 H P T / M E N 23 2.767 0.205 0.043 Non-HPT 43 3.245 0.526 0.079 Serum P T H ( a r b U l l ) Heal thy 98 0.75 0.17 0.02 HPT, S p o r a d i c 65 1.27 0.68 0.05 H P T /MEN 23 1.20 0.50 0.07 Non-HPT 43 0.80 0.25 0.04 155 T a b l e 2. Values in h e a l t h y s u b j e c t s s e p a r a t e d w i t h r e g a r d t o age a n d sex (Mean f SD) . AGE < 50 y e a r s > 50 y e a r s A l I (n = 50) ( n = 48) C a l ( m m o l I I ) Men 1.21 ? 0.05 1.20 f 0.06 1.21 f 0.05 Women 1.20 ? 0.04 1.21 f 0.04 1.20 f 0.04 C a T Men 2.45 f 0.07 Women 2.41 f 0.10 A l b u m i n ( g l l ) Men 44.2 f 2.9 Women 44.0 f 3.5 CaM Men 2.40 f 0.07 Women 2.38 ? 0.08 P T H ( a r b U / l ) Men 0.67 2 0.17 Women 0.82 f 0.18 - 2.40 f 0.08*) 2.42 f 0.09 42.2 f 3.3*) 41.9 f 1.8**) 2.40 f 0.09 2.43 f 0.08**) 0.71 i 0.14 0.85 f 0.14 2.43 f 0.07 2.40 f 0.09 43.5 f 3.2 43.0 f 3.0 2.40 k 0.07 2.40 f 0.08 0.68 t 0.16 0.83 f 0.16***) * ) p < 0.05 compared w i t h men < 5 0 y e a r s . * * ) p < 0.05 compared w i t h women < 50 y e a r s . * * * ) p < 0.001 compared w i t h males. R e l a t i o n s h i p s b e t w e e n C a l a n d CaM B o t h t h e h e a l t h y s u b j e c t s ( r = 0.33; p = 0.00 ) a n d t h e H P T p a t i - e n t s ( r = 0.90; p < O . O O l ) d i s p l a y e d highly s i g n i f i c a n t c o r r e l a t i o n s b e t - ween t h e v a l u e s f o r CaM a n d C a l . However, t h e slope f o r t h e r e g r e s - sion e q u a t i o n was s t e e p e r f o r t h e H P T p a t i e n t s t h a n f o r t h e h e a l t h y s u b j e c t s ( F i g . 2). T h e r e w e r e n o s i g n i f i c a n t d i f f e r e n c e s as r e g a r d s t h e r e l a t i o n s h i p s b e t w e e n CaM a n d C a l ( d a t a n o t s h o w n ) b e t w e e n H P T p a t i e n t s a n d those w i t h o t h e r causes o f hypercalcaemia . Within t h e r a n g e o f CaM v a l u e s b e t w e e n 2.45 a n d 2.75 m m o l l l t h e r e was n o s i g n i f i - c a n t c o r r e l a t i o n b e t w e e n t h e CaM a n d C a l v a l u e s f o r a n y o f t h e s t u d y g r o u p s (Fig. 3). 156 T a b l e 3. A v e r a g e t o t a l i n t r a - d i u r n a l v a r i a t i o n s in h e a l t h y s u b j e c t s ( n = 52), a n d p a t i e n t s w i t h h y p e r p a r a t h y r o i d i s m ( n = 50) a n d o t h e r hypercalcaernia ( n = 2)). H e a l t h y s u b j e c t s HPT Non-HPT H o u r Mean SD Mean SD Mean SD 08.00 12.00 16.00 08.00 12.00 16.00 08.00 12.00 16.00 08.00 12.00 16.00 08.00 12.00 16.00 C a l ( m m o l / l ) 1.15 0.045 1.15 0.042 1.14 0.051 CaT I m m o l / l ) 2.45 0.102 2.45 0.082 2.47 0.095 A l b u m i n (911) 42.1 3.1 42.9 2.6 42.9 3.3 CaM ( m m o l l l l 2.46 0.07 2.45 0.06 2.46 0.06 PTH ( a r b U / I 1 0.80 0.14 0.80 0.14 0.81 0.13 1.40 1.40 1.38 2.90 2.94 2.90 40.0 40.5 39.5 2.91 2.98 2.91 1.16 1.17 1.18 0.179 0.183 0.198 0.375 0.388 0.362 3.7 4.2 3.4 0.39 0.39 0.36 0.53 0.53 0.55 1.37 0.145 1.36 0.142 1.32 0.149 2.80 0.357 2.79 0.340 2.77 0.304 33.7 2.4 33.1 3.3 33.2 3.7 2.97 0.38 2.93 0.35 2.91 0.34 0.78 0.27 0.78 0.26 0.80 0.27 R e l a t i o n s h i p s between CaM a n d P T H T h e r e w e r e n o s i g n i f i c a n t c o r r e l a t i o n s between t h e CaM a n d P T H values e i t h e r in t h e g r o u p o f h e a l t h y s u b j e c t s ( r = 0.04) o r among t h o s e w i t h non-HPT hypercalcaemia ( F i g . 4). 157 T a b l e 4. A v e r a g e t o t a l i n t r a - i n d i v i d u a l v a r i a t i o n w i t h i n a n d between d a y s . W i t h i n - d a y Between-day (SD) (SD) C a l ( m m o l / l ) H e a l t h y 0.019 0.027 H P T 0.026 0.031 Non-HPT 0.041 0.060 CaT (rnmol/l) H ea I thy 0.043 0.032 H P T 0.054 0.071 Non-HPT 0.050 0.111 A l b u m i n ( g l l ) H e a l t h y 0.221 1.187 H P T 1.502 1.750 Non-HPT 0.992 1.110 CaM ( m m o l / l ) Heal thy 0.049 0.07 H P T 0.051 0.07 Non-HPT 0.063 0.12 P T H ( a r b U / I ) H e a l t h y 0.044 0.16 H P T 0.050 0.12 Non-HPT 0.045 0.05 When a l l t h e CaM v a l u e s f o r t h e HPT p a t i e n t s w e r e c o n s i d e r e d a highly s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n between CaM a n d P T H was e v i d e n t ( r = 0.62; pO. A. D i s c r i m i n a t i o n w i t h HPT (6) , 'A b b e t w e e n h e a l t h y s u b j e c t s ( A ) a n d p a t i e n t s C l a s s i f i e d g r o u p A B I. All Subjects a ) A l l s u b j e c t s ( C a M ) ' ) C o r r e c t g r o u p A 91 0 C o r r e c t g r o u p B 14 7 4 C o r r e c t g r o u p A 50 0 b ) Men ( & M I 2 ) C o r r e c t g r o u p B 1 15 c ) Women < 50 y e a r s (CaM, P T H ) 3 ) C o r r e c t q r o u p A 2 2 1 I . ' C o r r e c t g r o u p B 1 7 4 ) d ) Women > 5 0 y e a r s (CaM) C o r r e c t c l r o u p A 18 0 - . C o r r e c t g r o u p B 1 2 51 I I . S u b i e c t s w i t h CaM 2.45 - 2.75 mmol/l 5) a ) A l l s u b j e c t s [CaM, C a l ) C o r r e c t g r o u p B 1 31 b ) Men ( C a M j 6 ) C o r r e c t g r o u p A 2 9 1 C o r r e c t g r o u p A 1 6 0 C o r r e c t g r o u p 8 0 8 c ) Women < 50 y e a r s ( C a l , P T H ) 7 ) C o r r e c t g r o u p A 5 0 C o r r e c t g r o u p B 0 8 8 ) d ) Women > 50 y e a r s (CaM, C a l ) C o r r e c t g r o u p A 8 1 C o r r e c t g r o u p B 1 19 1 1 -878572 T A B L E 5. ( C o n t i n u e d ) . B. D i s c r i m i n a t i o n between p a t i e n t s w i t h HPT ( B ) a n d o t h e r causes o f hypercalcaemia ( C ) . A n i n d i v i d u a l i s allocated t o g r o u p B f o r zo. Classified g r o u p I. A l l Subiects a) A l l s u b j e c t s (CaM, Cal, P T H ) 1) C o r r e c t g r o u p B 74 14 b ) Men (CaM)21 C o r r e c t g r o u p C 7 14 C o r r e c t g r o u p C 12 34 C o r r e c t g r o u p B 14 3 c ) Women < 50 y e a r s ( P T H ) 3) C o r r e c t qrouI) B 7 1 C o r r e c t g r o u p c 0 5 d) Women > 50 y e a r s (CaM, PTH)‘) C o r r e c t g r o u p B 57 6 C o r r e c t g r o u p C 6 14 II. S u b i e c t s w i t h CaM 2.45 - 2.75 m m o l / l All s u b j e c t s (Car)’) C o r r e c t g r o u p B 22 10 C o r r e c t g r o u p c 2 5 1 ) z = 6.76xCaM-7.13xCal-3.34xPTH 2) 2 = 2.967 ( > = g r o u p C ) 3) 2 = 0.806 ( > = g r o u p B) 4) z = 4.OxCaM-3.72xPTH-8.64 5) = 1.293 ( > = g r o u p C ) 164 who h a d CaM v a l u e s below 2.75 mmol/I w e r e a n a l y z e d t h e c o m p u t e r p r o g - ramme o n l y selected C a l measurements t o separate t h e t w o g r o u p s most e f f i c i e n t l y ( T a b l e 5B 1 1 ) . Optimal d i s c r i m i n a t o r y levels o f CaM f o r s c r e e n i n g o f HPT In t h e r e f e r e n c e sample g r o u p o f h e a l t h y s u b j e c t s t h e d i s t r i b u t i o n o f CaM v a l u e s was close t o Gaussian as j u d g e d by s t a t i s t i c a l goodness-of f i t - t e s t s ( t h e Shapiro-Wilks t e s t f o r n < 50) a n d t h e Kolmogorov-Smirnow t e s t f o r n 2 50). In t h e H P T p a t i e n t g r o u p s , h o w e v e r , t h e fit was less close. Similar o b s e r v a t i o n s w e r e made f o r t h e C a l values. Since i t a p p e a r e d from t h e s e c a l c u l a t i o n s a n d p l o t s t h a t t h e f r e q u e n c y d i s t r i b u t i o n s f o r t h e H P T p a t i e n t s w e r e f a i r l y Gaussian o v e r t h e r i g h t - h a n d s i d e o f t h e c u r v e but n o t o n i t s e x t r e m e l e f t t h e f o l l o w i n g p r o c e d u r e was c a r r i e d o u t : T h e assembled CaM values w e r e r e o r g a n i z e d o n t h e a s s u m p t i o n t h a t t h e y c o n s t i t u t e d a p a r t o f a Gaussian d i s t r i b u t i o n , w h e r e t h e lowest v a l u e s h a d b e e n omitted. A t h e o r e t i c a l Gaussian d i s t r i b u t i o n f u n c t i o n was t h e n f i t t e d t o t h e t r u n c a t e d f r e q u e n c y d i s t r i b u t i o n giving estimates o f t h e location ( c ) a n d s t a n d a r d d e v i a t i o n (SD). F i g u r e 7 shows a Gaussian f u n c t i o n so f i t t e d u s i n g a n o n - l i n e a r p a r a - meter estimation p r o c e d u r e f o r t h e f r e q u e n c y d i s t r i b u t i o n s f o r CaM in t h e HPT p a t i e n t s . From t h i s f i g u r e it c a n b e seen t h a t t h e mean v a l u e a n d s t a n d a r d d e v i a t i o n (c = 2.81 f 0.23 m m o l / l ) f o r t h e t h e o r e t i c a l d i s t r i b u t i o n a r e somewhat l o w e r t h a n t h e c o r r e s p o n d i n g v a l u e o b t a i n e d in a s t r a i g h t f o r w a r d c a l c u l a t i o n o f t h e H P T p o p u l a t i o n (c = 2.91 * 0.30 m m o l / l ) . T h e optimal d i s c r i m a t o r y levels o f CaM w e r e c a l c u l a t e d f r o m t h e idealized c u r v e s as f u n c t i o n s o f w e i g h t i n g r a t i o s ( f a l s e p o s i t i v e o r false n e g a t i v e ) a n d f o r d i f f e r e n t p r e v a l e n c e s ( F i g . 8 ) . As c a n b e seen t h e optimal d i s c r i - m i n a t o r y l e v e l f o r a w e i g h t i n g r a t i o 1:l v a r i e d f r o m 2.73 mmol/l a t a p r e - valence o f 0.1 % t o 2.56 mmol/l w h e n t h e p r e v a l e n c e was assumed t o b e 50%. F u r t h e r m o r e , f o r a p r e v a l e n c e o f 5 0 % t h e optimal d i s c r i m i n a t o r y l i m i t was 2.63 mmol/l f o r a w e i g h t i n g r a t i o o f 1O:l f o r false p o s i t i v e t o false n e g a t i v e , but 2.47 mmol/l f o r t h e r e v e r s e w e i g h t i n g r a t i o . 165 Numbers ** *** **** 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 CaM (mmol/D F i g . 7. H i s t o g r a m f o r t h e a c t u a l v a l u e s o f CaM in p a t i e n t s w i t h h y p e r - p a r a t h y r o i d i s m . T h e f i t t e d t h e o r e t i c a l d i s t r i b u t i o n i s i n d i c a t e d by d o t t e d lines. 2.80 = 1 0 2.70 U - a, > a, - 2.60 f .- E 2.50 5 5 3 2.40 E 0 a C .#- .- v) .- .#- 0" h Prevalence 1 0.00 1 0.0 1 0.05 0.10 0.50 1O:l 5: 1 2: 1 1:l 1:2 1:5 1:lO Weighting ratio false positive/false negative Fig. 8 . T h e optimal d i s c r i m a t o r y level f o r s e r u m calcium (CaM) as a f u n c t i o n o f w e i g h t i n g - r a t i o false p o s i t i v e : false n e g a t i v e f o r d i f f e r e n t p r e v a l e n c e s o f h y p e r p a r a t h y r o i d i s m . 166 DISCUSS I O N T h e p r e s e n t s t u d y was c o n d u c t e d w i t h two major aims. T h e f i r s t was t o e s t a b l i s h t h e e x t e n t t o w h i c h measurements o f plasma i o n i z e d calcium a n d s e r u m PTH c o n t r i b u t e d t o t h e s e p a r a t i o n o f p a t i e n t s w i t h m i l d H P T f r o m h e a l t h y i n d i v i d u a l s w i t h CaM v a l u e s in t h e u p p e r p a r t o f t h e r e f e r e n c e r a n g e as well as in t h e d i f f e r e n t i a l d i a g n o s i s o f m a n i f e s t hypercalcemia. T h e o t h e r was t o fix t h e optimal d i s c r i m i n a t o r y l i m i t f o r s e r u m calcium in t h e d e l i n e a t i o n o f m i l d p r i m a r y H P T f r o m h e a l t h y s u b j e c t s in t h e u p p e r p a r t o f t h e r e f e r e n c e r a n g e . As a b a s i s f o r these c a l c u l a t i o n s i n f o r m a t i o n was c o l l e c t e d f o r assessment o f b o t h i n t r a - i n d i v i d u a l a n d i n t e r - d i u r n a l v a r i a t i o n s f o r t h e s t u d i e d v a r i - ables. D i u r n a l v a r i a t i o n s We f o u n d o n l y small, s t a t i s t i c a l l y i n s i g n i f i c a n t v a r i a t i o n s o f t h e s e r u m calcium a n d P T H c o n c e n t r a t i o n s t h r o u g h t h e d a y . A p e a k v a l u e f o r s e r u m calcium in t h e m o r n i n g h a s b e e n d e s c r i b e d (13.25) w h i l e a n ear,ly m o r n i n g n a d i r o f t o t a l calcium a n d i o n i z e d calcium has also b e e n r e c o r d e d (25,29). A d i u r n a l p a t t e r n o f s e r u m immunoreactive PTH has b e e n r e p o r t e d in b o t h normal s u b j e c t s a n d p a t i e n t s w i t h HPT (14,25,29,41) a n d also a r e l a - t i o n s h i p b e t w e e n P T H a n d sleep stages (27,39), f o u n d n o e v i d e n c e f o r r h y t h m i c e p i s o d i c v a r i a t i o n o f calcium o r PTH during t h e d a y . In t h e p r e s e n t s t u d y l i t t l e a t t e n t i o n was p a i d t o t h e i n f l u e n c e o f meals. A l t h o u g h it w o u l d b e o f some b e n e f i t t o h a v e a l l specimens f o r calcium a n d PTH t a k e n in t h e m o r n i n g w i t h t h e p a t i e n t f a s t i n g it seems t h a t f o r most p r a c t i c a l c l i n i c a l p u r p o s e s a random sample during t h e d a y i s s u f f i c i e n t . T h e i n t e r - d i u r n a l v a r i a t i o n s w e r e o n l y moderately g r e a t e r t h a n t h e i n t r a - d i u r n a l v a r i a t i o n s w h i c h w e r e c o m p a r a t i v e l y modest. T h e m a j o r i t y o f these v a r i a t i o n s c o u l d b e e x p l a i n e d by p r e - a n a l y t i c a l a n d a n a l y t i c a l v a r i a - t i o n s , i.e, t h e b i o l o g i c a l v a r i a t i o n s w e r e small. T h e 95 % c o n f i d e n c e i n t e r v a l f o r a s i n g l e s e r u m calcium v a l u e o f 2.50 mmol c o u l d b e c a l c u l a t e d t o b e 2.38 - 2.62 mmol/l. T h u s t h e c l i n i c a l r o u t i n e w h i c h r e q u i r e s s e v e r a l samples o n a l t e r n a t e d a y s m i g h t b e u n n e c e s s a r y in s u b j e c t s w h e r e t h e f i r s t v a l u e is lower t h a n 2.50 mmol /I. 167 A a e a n d sex v a r i a t i o n s T h e o b s e r v a t i o n o f h i g h e r CaM v a l u e s f o r e l d e r l y h e a l t h y women agrees w i t h r e c e n t f i n d i n g s in a l a r g e h e a l t h s u r v e y ( 3 6 ) . but i s in c o n t r a s t t o some e a r l i e r r e p o r t s w h e r e lower v a l u e s f o r t o t a l calcium in e l d e r l y p e r s o n s w e r e r e c o r d e d (15,26) o r n o c h a n g e w i t h age was o b s e r v e d . H o w e v e r , n o sex o r age d i f f e r e n c e was f o u n d f o r C a l in accordance w i t h p r e v i o u s r e p o r t s ( 4 2 ) . We did n o t find a n y s i g n i f i c a n t age-dependent d i f f e r e n c e s f o r t h e s e r u m P T H c o n c e n t r a t i o n s . H i g h e r P T H l e v e l s in o l d e r p e r s o n s h a v e p r e v i o u s l y b e e n r e p o r t e d ( 1 0,16,22,34,47). However, M a r c u s e t al . ( 3 4 ) r e p o r t e d t h a t w h e n t h e v a l u e s w e r e c o r r e c t e d f o r g l o m e r u l a r f i l t r a t i o n r a t e a d i f f e r e n c e r e l a t e d t o age was n o t s i g n i f i c a n t in h e a l t h y s u b j e c t s . C l e a r l y s i g n i f i c a n t s e x d i f f e r e n c e s w e r e n o t e d in t h e p r e s e n t s t u d y , f o r s e r u m PTH w i t h a 20 % h i g h e r values in females. Similar f i n d i n g s h a v e n o t p r e v i o u s l y b e e n g e n e r a l l y o b s e r v e d ( 3 0 ) . T h e reasons f o r t h e s e x d i f f e r - ences a r e n o t o b v i o u s s i n c e t h e y r e l a t e t o b o t h p r e - a n d postmenopausal females. A n a p p a r e n t c l i n i c a l consequence o f t h e o b s e r v a t i o n s t h a t b o t h CaM a n d P T H a r e h i g h e r in h e a l t h y post-menopausal females i s t h a t t h e r e w i l l b e a n i n c r e a s e d n u m b e r o f diagnosed H P T in t h i s g r o u p i f i d e n t i c a l c u t - o f f p o i n t s a r e u s e d as in males a n d y o u n g e r females, g i v e n t h e e x i s t e n c e o f a n u m b e r o f p a t i e n t s w i t h m i l d H P T who o t h e r w i s e remain u n r e c o g n i z e d . T h i s m i g h t t o some e x t e n t e x p l a i n t h e a p p a r e n t l y h i g h e r p r e v a l e n c e o f H P T in postmenopausal women ( 1 1 1. D e l i n e a t i o n o f HPT T h e a v a i l a b i l i t y o f measurements o f plasma i o n i z e d c a l c i u m a n d s e r u m PTH in c l i n i c a l p r a c t i c e has a r o u s e d g r e a t e x p e c t a t i o n s f o r i m p r o v e d d i a g - n o s i s o f HPT, r e g a r d i n g b o t h t h e d i f f e r e n t i a l d i a g n o s i s o f hypercalcemia a n d t h e s e p a r a t i o n f r o m t h e u p p e r r e f e r e n c e r a n g e o f h e a l t h y s u b j e c t s . S e v e r a l s t u d i e s h a v e d e s c r i b e d cases w h e r e a n e l e v a t e d plasma ionized calcium was f o u n d t o g e t h e r w i t h a v a l u e w i t h i n t h e r e f e r e n c e r a n g e f o r t o t a l s e r u m calcium, a n d w h e r e s u b s e q u e n t n e c k e x p l o r a t i o n r e v e a l e d HPT (7,23,32). I t seems logical t h a t in some p a t i e n t s p r o t e i n - b i n d i n g o f f r e e calcium v a r i e s so t h a t t h e t o t a l s e r u m calcium v a l u e m i g h t b e misleading ( 3 7 ) . However, if ionized calcium i s s u b s t i t u t e d f o r t o t a l s e r u m calcium mea- s u r e m e n t s in t h e c l i n i c a l r o u t i n e it seems l i k e l y t h a t a n o v e r l a p w i l l o c c u r b e t w e e n t h e u p p e r p a r t o f t h e d i s t r i b u t i o n o f t h e r e f e r e n c e v a l u e s o f 168 h e a l t h y i n d i v i d u a l s a n d t h e l o w e r p a r t o f t h e c o r r e s p o n d i n g d i s t r i b u t i o n c u r v e f o r p a t i e n t s w i t h HPT. A t least in t h e p r e s e n t s t u d y t h e o v e r l a p b e t w e e n t h e t w o p o p u l a t i o n s was o f a similar m a g n i t u d e as r e g a r d s i o n i z e d calcium v a l u e s a n d t o t a l , a l b u m i n - c o r r e c t e d values. T h e t e c h n i q u e s for measurement o f t h e free, i o n i z e d , f r a c t i o n o f s e r u m calcium h a v e o n l y r e c e n t l y become a v a i l a b l e f o r g e n e r a l c l i n i c a l r o u t i n e use. Many e a r l i e r s t u d i e s w e r e t h e r e f o r e p e r f o r m e d f o r t h e p u r p o s e o f d e v e l o p i n g f o r m u l a s w h e r e t h i s f r a c t i o n c o u l d b e estimated f r o m measurements o f t h e t o t a l s e r u m calcium a n d c a l c i u m - b i n d i n g p r o t e i n s , p r i m a r i l y a l b u m i n (12,31,35,38). In c l i n i c a l p r a c t i c e it has g e n e r a l l y b e e n c o n s i d e r e d con- v e n i e n t t o a d j u s t t h e s e r u m calcium a c c o r d i n g t o t h e v a l u e o f t h e s e r u m a l b u m i n from t h e r e f e r e n c e mean v a l u e . S u c h a c o r r e c t i o n has been demon- s t r a t e d t o i m p r o v e t h e d i a g n o s i s o f HPT ( 4 3 ) . We f o u n d , as B r a u m a n e t al. ( 6 ) t h a t t h e slope f o r i o n i z e d calcium o n t o t a l ( m o d i f i e d ) s e r u m calcium was steeper f o r t h e hypercalcaemic p a t i e n t s t h a n f o r t h e h e a l t h y c o n t r o l s , i.e. t h e p a t h o l o g i c a l c o n d i t i o n leading t o a r i s e in t h e plasma i o n i z e d calcium, did n o t p r o p o r t i o n a l l y i n c r e a s e t h e f r a c t i o n b o u n d t o albumin. P a t i e n t s w i t h HPT c o n s e q u e n t l y h a d h i g h e r values f o r C a l , f o r c o r r e s p o n d i n g v a l u e s o f CaM. Measurements o f PTH did n o t completely s e p a r a t e p a t i e n t s w i t h m i l d H P T f r o m h e a l t h y s u b j e c t s . T h e r e i s a c o r r e l a t i o n b e t w e e n t h e g l a n d u l a r mass, s e r u m P T H a n d s e r u m calcium in p a t i e n t s w i t h p r i m a r y HPT. T h u s p a t i e n t s w i t h t h e m i l d e s t hypercalcemia h a v e t h e smallest g l a n d s , o f t e n w i t h modest h y p e r p l a s i a , a n d t h e y also h a v e s e r u m PTH values close t o or e v e n w i t h i n t h e r e f e r e n c e r a n g e of h e a l t h y i n d i v i d u a l s ( 5 0 ) . F u r t h e r m o r e , a t t h e c e l l u - l a r l e v e l p a t i e n t s w i t h t h e m i l d e s t h y p e r c a l c e m i a h a v e a close t o normal r e s p o n s e t o a l t e r a t i o n s o f t h e a m b i e n t calcium c o n c e n t r a t i o n s as r e g a r d s t h e release o f P T H (40.45). E v a l u a t i o n s o f s e r u m PTH c o n c e n t r a t i o n s m u s t t a k e i n t o c o n s i d e r a t i o n t h e f a c t s t h a t s e v e r a l f r a g m e n t s o f P T H a r e c i r c u l a t i n g , a n d t h a t t h e r e l a t i v e amount o f d i f f e r e n t f r a g m e n t s may b e a l t e r e d by v a r i o u s d i s o r d e r s (1,4,19). T h e c l i n i c a l u s e f u l n e s s o f each assay m u s t t h e r e f o r e b e e s t a b l i s h e d e m p i r i c - a l l y ( 7 ) . I T h e a n t i s e r u m u s e d in t h e p r e s e n t s t u d y has a high a f f i n i t y f o r i n t a c t PTH. A s t h e r e was a f a i r l y r a p i d e l i m i n a t i o n o f immunoreactive hormone from t h e c i r c u l a t i o n in p a t i e n t s o p e r a t e d f o r HPT ( F i g . 1 ) it seems u n l i k e l y t h a t f r a g m e n t s w i t h a slow e l i m i n a t i n g r a t e s t r o n g l y i n f l u e n c e d t h e assay. 169 P r e v i o u s i n v e s t i g a t i o n s w i t h t h e same assay h a v e in p a t i e n t s w i t h i m p a i r e d r e n a l f u n c t i o n d e m o n s t r a t e d a close c o r r e l a t i o n between s e r u m levels o f P T H , b o n e r e s o r p t i o n s u r f a c e s a n d bone f o r m a t i o n r a t e s ( 3 4 ) . A r a p i d increase in t h e PTH response t o l o w e r i n g o f s e r u m calcium w i t h E D T A has also b e e n o b s e r v e d ( 3 ) . A l l t h e s e o b s e r v a t i o n s i n d i c a t e t h a t t h e assay d e t e c t s m a i n l y i n t a c t PTH a n d o n l y t o a lesser e x t e n t c a r b o x y l - t e r m i n a l f r a g m e n t s o f P T H w h i c h a r e k n o w n t o b e s l o w l y eliminated f r o m t h e c i r c u l a t i o n . I t has been r e p o r t e d t h a t assay system d e t e c t i n g t h e C - t e r m i n a l r e g i o n o f P T H h a v e a g r e a t e r c a p a c i t y t o d i s c l o s e p r i m a r y H P T o n basal mea- surements o f PTH t h a n d o those system w h i c h a r e n o t capable o f d e t e c t i n g r a p i d changes in s e c r e t i o n ( 8 ) . From d e t e r m i n a t i o n s o f P T H alone less t h a n h a l f o f a l l p a t i e n t s w i t h H P T were c l e a r l y s e p a r a t e d f r o m t h e h e a l t h y s u b - j e c t s a n d t h e s e p a r a t i o n a g a i n s t o t h e r causes o f hypercalcaemia was less t h a n 75 %. When t h e simultaneously measured calcium c o n c e n t r a t i o n s w e r e also c o n s i d e r e d b e t t e r s e p a r a t i o n s w e r e o b t a i n e d , but t h e d e l i n e a t i o n s w e r e still n o t complete, p a r t i c u l a r l y in t h o s e w i t h m i l d hypercalcaemia. P T H i s p r i m a r i l y eliminated t h r o u g h g l o m e r u l a r f i l t r a t i o n , a n d i m p a i r e d r e n a l f u n c t i o n w i l l t h e r e f o r e i n a p p r o p r i a t e l y r a i s e t h e PTH levels. S u c h a r i s e in P T H l e v e l s i s more p r o m i n e n t w i t h assays d i r e c t e d t o w a r d s t h e c a r b o x y - t e r m i n a l p a r t o f t h e hormone. In g e n e r a l , w i t h o u r assay system moderately i m p a i r e d r e n a l f u n c t i o n , w i t h o u t s e c o n d a r y h y p e r p a r a t h y r o i d i s m , does n o t r a i s e t h e PTH levels above t h e normal r a n g e ( 3 4 ) . In p a t i e n t s w i t h hypercalcaemia o f o t h e r o r i g i n t h a n H P T r e n a l f u n c t i o n i s o f t e n i m p a i r e d ( 3 3 ) a n d t h e s e r u m c o n c e n t r a t i o n s o f P T H may t h e r e f o r e b e e l e v a t e d e v e n above t h e r e f e r e n c e r a n g e a l t h o u g h t h e s e c r e t i o n o f P T H i s in f a c t s u p p - r e s s e d by t h e hypercalcaemia. A moderate r e d u c t i o n o f g l o m e r u l a r f i l t r a t i o n , as e v i d e n c e d by a n i n c r e a s e o f t h e s e r u m c r e a t i n i n e v a l u e s , was p r e s e n t in s e v e r a l o f o u r p a t i e n t s w i t h n o n - H P T hypercalcaemia, a f a c t w h i c h m i g h t e x p l a i n some o f t h e o v e r l a p between t h e P T H v a l u e s b e t w e e n t h e h y p e r - calcaemic p a t i e n t g r o u p s . One p a t i e n t , w i t h sarcoidosis, w i t h a serum c r e a t i n i n e v a l u e a r o u n d 300 umol/I e v e n h a d a m a r k e d l y e l e v a t e d P T H v a l u e ( 2 . 2 a r b U / l ) . W i t h o u t p e r f o r m i n g n e c k e x p l o r a t i o n in all hypercalcaemic p a t i e n t s it c o u l d n o t b e d e f i n i t e l y e x c l u d e d t h a t some o f t h e m did n o t also h a v e HPT. P a t i e n t s w i t h o u t m a l i g n a n t d i s o r d e r s , h o w e v e r , d i s p l a y e d n o r m a l i z a t i o n o f t h e i r hypercalcaemia w h e n t h e i r u n d e r l y i n g disease r e c e d e d ( s a r c o i d o s i s , 170 t h y r o t o x i c o s i s , immobilisation) a n d also in most o f t h o s e w i t h malignancies t h e c l i n i c a l p i c t u r e was c h a r a c t e r i s t i c o f hypercalcaemia o f o t h e r o r i g i n t h a n HPT. When t h e c l i n i c a l e x p e r i e n c e s o f v a r i o u s r e s e a r c h c e n t e r s a r e compared t h e i n v e s t i g a t e d p a t i e n t p o p u l a t i o n s m u s t also b e c o n s i d e r e d . A t o u r h o s p i - t a l , we h a v e f o r s e v e r a l y e a r s b e e n i n t e r e s t e d in p a t i e n t s w i t h m i l d h y p e r - calcaemia a n d also h a d a l i b e r a l a t t i t u d e t o w a r d s s u r g i c a l t r e a t m e n t o f p a t i e n t s w i t h s u s p e c t e d m i l d HPT. In o u r c o n s e c u t i v e s e r i e s o f p a t i e n t s t h e r e a r e t h e r e f o r e many b o r d e r l i n e s e r u m calcium v a l u e s . T h e d i s c o v e r y o f a g r e a t o v e r l a p o f PTH values b e t w e e n t h e p a t i e n t s w i t h m i l d H P T a n d t h e h e a l t h y r e f e r e n c e p o p u l a t i o n s h o u l d b e r e g a r d e d in t h i s light. If o n l y p a t i e n t s w i t h high s e r u m calcium v a l u e s , above 2 . 9 0 m m o l / l , h a d been con- s i d e r e d as h a v i n g HPT, t h e d i a g n o s t i c d i f f i c u l t i e s w i t h r e g a r d t o b o t h s e p a r a t i o n t o w a r d s t h e h e a l t h y s u b j e c t s a s well as a g a i n s t o t h e r causes o f hypercalcaemia w o u l d h a v e been m u c h less t h a n we now e x p e r i e n c e d . D i s c r i m i n a t o r v a n a l v s i s T h e d i s c r i m i n a t o r y a n a l y s i s was c a r r i e d o u t in t w o s t e p s , i n c l u d i n g f i r s t a l l i n d i v i d u a l s in t h e t w o g r o u p s t o b e compared. In a second s t e p o n l y those w i t h b o r d e r l i n e CaM v a l u e s ( i . e . 2 . 4 5 - 2 . 7 5 m m o l / l ) w e r e c o n s i d e r e d . T h e r a t i o n a l e f o r t h e l a t t e r r e s t r i c t i o n was t h a t o t h e r w i s e p a t i e n t s w i t h t h e most abnormal biochemical d e v i a t i o n s w o u l d a f f e c t t h e d i s c r i m i n a t o r y f u n c t i o n o u t o f p r o p o r t i o n t o t h e d e s i r e d goal. T h e c l i n i c a l p r o b l e m , o b v i o u s l y , does n o t c o n s i s t in s e p a r a t i n g a H P T p a t i e n t w i t h a s e r u m calcium v a l u e o f 3 . 5 - 4 m m o l / l f r o m t h e h e a l t h y p o p u l a t i o n but r a t h e r t o d i s c l o s e t h e m i l d e s t f o r m o f HPT. In t h e s t a t i s t i c a l analyses it was a p p a r e n t t h a t measurements o f CaM alone c o u l d s e p a r a t e t h e m a j o r i t y o f H P T p a t i e n t s f r o m t h e h e a l t h y s u b j e c t s . T h i s i s almost s e l f - e x p l a n a t o r y as t h e m a j o r i t y o f H P T p a t i e n t s h a v e c l e a r - c u t hypercalcaemia. In s u c h i n s t a n c e s a d d i t i o n a l measurements ( o f C a l o r P T H ) c a n n o t allow o f f u r t h e r s e p a r a t i o n . However, w h e n o n l y t h e b o r d e r l i n e i n d i v i d u a l s w e r e a n a l y z e d Cal p r o v e d t o b e a u s e f u l a d d i t i o n a l determina- t i o n . A l t h o u g h t h e r e was a g e n e r a l c o r r e l a t i o n b e t w e e n CaM [ a n d C a l l a n d PTH f o r a l l t h e H P T p a t i e n t s t h e m i l d p a r a t h y r o i d h y p e r f u n c t i o n in those w i t h t h e m i l d e s t hypercalcaemia did n o t r e s u l t in s i g n i f i c a n t e l e v a t i o n s o f t h e P T H l e v e l s a n d in t h i s area P T H measurements did n o t p r o v i d e a n y f u r t h e r d i s c r i m i n a t i o n . 171 A l t h o u g h t h e r e w e r e some age a n d sex d i f f e r e n c e s f o r b o t h CaM a n d P T H these w e r e a p p a r e n t l y n o t s u c h as t o i m p r o v e t h e d i s c r i m i n a t i o n in t h e p r e s e n t material. F o r t h e d i f f e r e n t i a l d i a g n o s i s o f hypercalcaemia it t u r n e d o u t t h a t measurements o f P T H w e r e v a l u a b l e , p a r t i c u l a r l y in i n d i v i d u a l s w i t h t h e h i g h e s t s e r u m calcium values. In a d d i t i o n C a l v a l u e s w e r e h i g h e r f o r t h e H P T p a t i e n t s t h a n f o r t h e non-HPT hypercalcaemics, p a r t i c u l a r l y w i t h i n t h e l o w e r r a n g e o f r a i s e d s e r u m calcium values. T h i s i s most l i k e l y e x p l a i n e d by a n " o v e r - c o r r e c t i o n " o f t h e CaM values f o r t h e decreased s e r u m a l b u m i n , o f t e n f o u n d in p a t i e n t s w i t h hypercalcaemia ( 5 ) . Optimal d i s c r i m i n a t o r y l i m i t B e f o r e c a l c u l a t i o n o f t h e optimal d i s c r i m i n a t o r y l i m i t s b e t w e e n t h e h e a l t h y s u b j e c t s a n d p a t i e n t s w i t h m i l d H P T a n a d j u s t m e n t o f t h e f r e q u e n c y d i s t r i - b u t i o n c u r v e for CaM in t h e H P T p a t i e n t s was p e r f o r m e d . T h e reason f o r t h i s m a n o e u v r e was t h e o b s e r v a t i o n t h a t t h e calcium v a l u e s a p p r o a c h e d a Gaussian d i s t r i b u t i o n e x c e p t in t h e lower p a r t w h e r e t h e r e was a l a c k o f v a l u e s below 2.60 m m o l l l t o achieve a b e l l - s h a p e d c u r v e . T h e c o n c e p t o f llnormocalcemic p r i m a r y H P T " has b e e n d i s c u s s e d f o r s e v e r a l y e a r s (23,46,48). Many p a t i e n t s r e p o r t e d t o h a v e normocalcemic p r i m a r y H P T a p p e a r t o h a v e H P T w i t h a m i l d , sometimes f l u c t u a t i n g , h y p e r - calcemia (24,28). H o w e v e r , s e v e r a l o b s e r v a t i o n s i n d i c a t e t h a t H P T c o u l d e x i s t also in p a t i e n t s who n e v e r d i s p l a y r a i s e d s e r u m calcium values. F o r i n s t a n c e s e r i e s o f p a t i e n t s h a v e b e e n p r e s e n t e d w h e r e n e c k e x p l o r a t i o n h a s b e e n c a r r i e d o u t d e s p i t e c o n s t a n t normocalcemia (17.23) a n d w h e r e d e f i n i t e H P T h a s b e e n c o n f i r m e d h i s t o p a t h o l o g i c a l l y . In a r e c e n t s e r i e s a p p r o x i m a t e l y 40 p a t i e n t s o p e r a t e d a t o u r h o s p i t a l h a d s e r u m calcium v a l u e s b e t w e e n 2.60 a n d 2.80 mmol/l. HPT c o u l d b e o p e r a t i v e l y v e r i f i e d in a l l p a t i e n t s ( 5 0 ) . T h i s s u g g e s t s t h a t some i n d i v i d u a l s w i t h s e r u m v a l u e s below 2.60 m m o l / l also h a v e HPT. T h i s d i s c u s s i o n o f "normocalcemic H P T I' o n l y s e r v e s as a n e x p l a n a t i o n o f t h e mathematical a n a l y s i s a n d does n o t c a r r y a n y i m p l i c a t i o n s as t o w h e t h e r s u c h p a t i e n t s s h o u l d b e s o u g h t o r e v e n less w h i c h t r e a t m e n t i s optimal. O n t h e b a s i s o f t h e s e r e s u l t s we c o u l d c a l c u l a t e a n optimal d i s c r i m i n a t o r y l i m i t f o r H P T o f 2.68 m m o l l l , assuming a p r e v a l e n c e of 1% a n d a w e i g h t i n g r a t i o o f 1:l b e t w e e n f a l s e p o s i t i v e s a n d false n e g a t i v e s . A s m e n t i o n e d 172 above, h o w e v e r , o n l y p o s i t i v e e x p l o r a t i o n s w e r e p e r f o r m e d in c o n s e c u t i v e p a t i e n t s w i t h s u c h calcium values. If o t h e r p r e r e q u i s i t e s a r e c o r r e c t , t h i s finding i n d i c a t e s t h a t t h e p r e v a l e n c e o f H P T in a g e n e r a l p o p u l a t i o n m u s t b e c o n s i d e r a b l y h i g h e r t h a n 1%. In s u p p o r t of t h i s v i e w , a u t o p s y s t u d i e s h a v e f o u n d adenomatous H P T in as many as 2.4 % o f i n d i v i d u a l s above t h e age o f 70, a n d e v i d e n c e of h y p e r p l a s t i c p r i m a r y H P T in f u r t h e r 7 % ( 5 0 ) . From Fig. 8 it c a n b e seen t h a t t h e w e i g h t i n g r a t i o o f 1 : l (i.e. e q u a l i m p o r t a n c e i s p a i d t o false p o s i t i v e a n d false n e g a t i v e c l a s s i f i c a t i o n s ) f o r a s e r u m calcium v a l u e of 2.60 m m o l / l c o r r e s p o n d s t o a p r e v a l e n c e in t h e p o p u l a t i o n o f a b o v e 10 %. F i r s t d e g r e e r e l a t i v e s o f p a t i e n t s w i t h t h e MEN-1 s y n d r o m e h a v e a 50 % r i s k o f b e i n g c a r r i e r s o f t h e MEN-1 t r a i t . In a p o p u l a t i o n w i t h a 50 % p r e v a l e n c e o f H P T t h e optimal d i s c r i m i n a t o r y l i m i t f o r s e r u m calcium in t h e p r e s e n t s t u d y was 2.56 mmol/l i f it was c o n s i d e r e d e q u a l l y i m p o r t a n t t o a v o i d f a l s e l y p o s i t i v e a n d falsely n e g a t i v e c l a s s i f i c a t i o n s . It i s a moot p o i n t w h e t h e r t h e w e i g h t i n g r a t i o in t h i s p a r t i c u l a r s e t t i n g i s d i f f e r e n t from o t h e r s i t u a t i o n s w h e r e H P T i s suspected. Whatever s t a n d p o i n t i s t a k e n in t h e i n d i v i d u a l c l i n i c a l s i t u a t i o n it i s a p p a r e n t f r o m these c o n s i d e r a t i o n s t h a t in MEN-1 r e l a t i v e s s e r u m calcium v a l u e s e v e n w i t h i n t h e r e f e r e n c e r a n g e m i g h t b e compatible w i t h H P T a n d t h e r e f o r e necessitate f u r t h e r i n v e s t i g a t i o n s . ACKNOWLEDGEMENT T h i s s t u d y was s u p p o r t e d by t h e S w e d i s h Medical Research C o u n c i l . REFERENCES 1. 2. 3 . 4. 5. A r n a u d , C.D., Goldsmith, R.S., B o r d i e r , P.J., Sizemore, G.W., L a r s e n , J.A. & G i l k i n s o n , J.: I n f l u e n c e o f immunoheterogeneity o f c i r c u l a t i n g p a r a t h y r o i d hormone o n t h e r e s u l t s of radioimmunoassays o f s e r u m in man. Am J Med 56:785-793, 1974. 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