Upsala J Med Sci 87: 151-161, 1982 Thermodynamical Aspects on the Determination of Bicarbonate in Urine Morgan Sohtell and Bertil Karlmark depart men^ of Physio/ogy and Medical Biophysics. Uppsala University, Uppsalu, Sweden ABSTRACT Despite t h e fact that the composition o f urine varies a l o t during the day, this has essentially been neglected as a f a c t o r o f importance in determinations o f urine bicarbonate. The investigations reviewed in combination w i t h an own study shows t h a t qualitative and quantitative factors in urine composition impacts the solubility o f carbon dioxide as w e l l as the dissociation constant f o r the bicarbonate b u f f e r system. These t w o "constants" are o f outstanding importance in the determination o f bicarbonate, using the t o t a l carbonic acid method as w e l l as the carbon dioxide equilibration method. Nomograms are presented t o quantify the influence o f d i f f e r e n t urine compositions on the determinations o f bicarbonate in f i n a l urine and tubular fluid. INTRODUCTION Since many decades it has been a well-known f a c t t h a t the ligands used i n the chemical description o f biological acid-base equilibria are influenced by d i f f e r e n t physical and chemical properties of the solution under study. F o r many biological fluids this f a c t has been made, f o r example, f o r blood (27), f o r cerebrospinal f l u i d (24, 25), f o r amniotic f l u i d (17) and also for a r t i f i c i a l fluids ( 9 , 32). A common denominator f o r these biological fluids i s the r e l a t i v e l y r e s t r i c t e d variation in composition f r o m t i m e t o t i m e and also between d i f f e r e n t individuals. As a sharp contrast, f i n a l urine varies in composition considerably even f r o m hour t o hour in t h e same individual. The circadian pH-variation (15) i s a well-known f a c t as w e l l as the postprandial "alkalaine tide". B u t the wide range in e l e c t r o l y t e composition and osmolality, makes a considerable i m p a c t on the chemical analysis o f components in the bicarbonate b u f f e r system. 151 Consider t w o f i n a l urine samples f r o m the same individual , the same day; one o f them 150 mOsm/kg and t h e other 600 mOsm/kg. Provided the osmolality r e f l e c t s the N a C l content the bicarbonate a c t i v i t i e s can be calculated using the Henderson-Hasselbalch equation (see Table 1). Thus, despite the f a c t t h a t the p H and Pco2 are identical i n the same samples, the bicarbonate a c t i v i t y measured m i g h t vary around 50% depending upon chemical factors other than the bicarbonate a c t i v i t y itself. 150 mOsm/kg 600 mOsm/kg PKa 6.117 5.919 S 0.03145 0.02915 H C O j 6 9 TABLE 1 Fitzsimons and Sendroy, ( 9 ) Van Slyke e t al., (32) Table 1.The influence o f osmolarity on the bicarbonate a c t i v i t y in urine. The calculations are based on a Pcop o f 40 m m H g (5.3 kPa) and a p H o f 6.8 f o r both urines. I n biological fluids other than urines, the bicarbonate variations as a function o f thermodynamical ligands is smaller and usually well-known (for a recent review see Siggaard Andersen, (27)). The a i m o f the present paper i s t o present ligands f o r the bicarbonate determination in urine as a function o f d i f f e r e n t compositions o f this fluid. A review o f the l i t e r a t u r e i n this f i e l d i s presented as w e l l as a study o f primary urine (ultrafiltrate). A n evaluation o f d i f f e r e n t methods f o r the bicarbonate determination i n urine i s made. MET H 0 D S The bicarbonate concentration can be analyzed i n d i f f e r e n t ways among which the determination o f t o t a l carbonic acid, Tcop is the most widely used. This technique cannot, however, d i f f e r between CO2, H C O j and CO;-, which i s a considerable drawback when dealing w i t h fluids o f unknown p H and CO2 concentrations. This is most times t h e case i n compartment studies i n or adjacent t o single cells. Pcop electrodes are now made f o r in vivo studies in micropuncture research (8,30), b u t w i l l probably w a i t a couple of years f o r a more wide-spread use. The bicarbonate a c t i v i t i e s in biological fluids are determined mainly by using the equilibration technique as thoroughly used and described f o r blood and other fluids 152 (27). This technique i s also suitable f o r samples i n nano-liter size as described by K a r l m a r k and Sohtell (18). For carbonic anhydrase r i c h fluids (eg blood), chemical equilibrium i s assumed and the calculation o f bicarbonate a c t i v i t y is based upon single measurements of p H and Pco2. If chemical non-equilibrium (disequilibrium) i s prevailing, the Henderson-Hasselbalch equation cannot be used. I n such a case an immediate responding and highly specific bicarbonate electrode i s the only method. Such an electrode is described (19) but i s not yet developed enough t o p e r m i t reliable and valid measurements. As long as the non-bicarbonate b u f f e r concentration i s low as compared t o the bicarbonate a c t i v i t y the equilibration technique i s rather insensitive t o differences in actual p H and Pco2 and i s preferred mainly because i t s s i m p l i c i t y and also the f a c t t h a t it results i n a measure of the a c t i v i t y and not barely by the concentration. The t o t a l - CO2 technique f o r bicarbonate determinations gives a more unspecific t o t a l concentration o f bicarbonate buffer constituents and gives a considerable error i n acid urines. Irrespective o f which o f the mentioned techniques that is t o be used f o r bicarbonate determinations i n urine, the CO2 concentration must be measured. This i s easily performed w i t h a Pco2 electrode as long as the sample volume i s loop1 or more. For t h e calculation of the bicarbonate a c t i v i t y t h e solubility f a c t o r ( S ) and pKa are then o f v i t a l importance. I n addition, knowledge o f the actual p H i s also v i t a l i n very alkaline urines (where the amount o f CO$- gives a substantial contribution ( 5 - 10 Yo ) t o totalLC02) and also i n highly buffered urine. So f a r experimental investigations o f the S and pKa f o r urines are not published. We used here one method described by Siesjo (24) t o study S and another by Siesjo (25) t o study pKa. P r i m a r y urine was a r t i f i c i a l l y made as an u l t r a f i l t r a t e of r a t plasma, f i l t e r e d through a D i a f l o membrane (PM 10, Amicon Corp., Lexington, Mass., USA). a)The solubility coefficient (S): The solution investigated was acidified w i t h HCI t o a p H of about 2.5 and then equilibrated w i t h the humidified 5 YO CO2 in oxygen gas m i x t u r e for 1 hour a t 38OC. The carbonic acid content of an equilibrated sample was made volatile by depositing it in a H C I solution i n a Conway unit, which was modified as shown i n Fig.1. The CO2 formed was trapped in a Ba(OH)2 solution. The sealing between the plugs and t h e l i d was made by the use o f acidified carboxymethylcellulose t o avoid leakage o f CO2. The diffusion time was 90 m i n and the change o f the Ba(OH)2 solution due t o the reaction w i t h CO2 was immediately analyzed by t i t r a t i o n w i t h H C I standard. S was measured as a relation between the t o t a l carbonic acid content (Tco2) i n acidified solution and the p a r t i a l pressure o f carbon dioxide. The validity o f the method was tested by determining the S f a c t o r f o r distilled water and a 160 m m o l f l N a C l solution. 153 H C1 \ HCl + sample Fig.1. The circular Conway unit (made of glass), h e r e seen from t h e side. The plugs a r e s e a l e d t o t h e lid with acidified carboxymethylcellulose. b) The f i r s t a p p a r e n t dissociation c o n s t a n t (pKa): The solution was f i r s t equlilibrated in a humidified g a s of 5% C O P in oxygen f o r one hour a t 38OC. The pH a t this equilibration w a s measured with a glass e l e c t r o d e and t h e P c o 2 w a s measured as described above. This value was inserted in t h e Henderson-Hasselbalch equation. RESULTS AND DISCUSSION a) The solubility coefficient: The r e s u l t s a r e presented a t t h e bottom of Table 2. Van Slyke et a1 (32) presented d a t a , indicating t h a t not only t h e ionic s t r e n g t h was of i m p o r t a n c e f o r t h e C O 2 solubility. Of considerable i m p o r t a n c e was also t h e influence of t h e d i f f e r e n t ionic species. The solubility of C O P is depressed by ions in proportion t o t h e i r c o n c e n t r a t i o n s (Table 3 ) . The t a b l e shows t h e depression of t h e solubility of CO2 f o r d i f f e r e n t ions e x t r a p o l a t e d t o a c o n c e n t r a t i o n of 1 mol/l. In lower c o n c e n t r a t i o n s t h e depression in solubility is proportionally reduced (for phosphate t h i s linearity is not s t r i c t , however). I t must be pointed o u t t h a t t h e t a b l e is not useable f o r c o n c e n t r a t i o n s above 300 mmol/l of individual ions. For biological pure s a l t solutions, Siggaard Andersen (27), summarized t h e d i f f e r e n t d a t a of t a b l e 3 in a formula, which describes an e s t i m a t i o n of t h e ionic influence but in t e r m s of a weighed ionic s t r e n g t h . But his approximation is not valid f o r urines, due t o t h e wide range of ionic composition during d i f f e r e n t physiological d i u r e t i c conditions. 154 Table 2 S 0.03304 0.03222 0.03015 0.03215 0.03007 0.0 3 2 6 2 0.03229 0.03136 0.03105 0.03223 0.03013 0.0311 0.03074 0.03065 0.03233 OC Solution 38 H 2 0 38 H20 38 Plasma (human) 38 H20 38 Plasma (ox) 37.5 H20 38 H20 * 37.5 160 m m o l / l N a C l 38 160 m m o l / l N a C l * 38 H20 38 Sera (human) 37 Amnion f l u i d (human) 38 U l t r a f i l t r a t e 38 160 m m o l / l N a C l 38 H20-"- Bohr,C., (4) Van Slyke e t al., (32) -II- Bartels and Wrbitzky, ( 2 ) - 1 8 - Siesjo, (24) -11- -11- -11- Austin e t al., (1) -11- Johnell, (17) This study (SE= 0.0018,n= 24) -'I- (SE= 0.00004,n=30) -11- (SE= 0.00009,n=47) Table 2. Summary o f data concerning the solubility o f COP in d i f f e r e n t kinds of solutions. In case the solubility was presented as the Bunsen coefficient ( l i t e r gas dissolved / l i t e r / u n i t atmosphere pressure), we have normalized it i n t o the solubility coefficients (mmol/l/mm Hg). Those values presented w i t h a * are a temperature correction t o 380 C f r o m the preceding value (see text). Table 3 H+ H C 2 0 4 L a c t a t e - c1- K+ Na+ H2POi HCOJ* 0.0 0 0 0 0 0.00117 0.00296 0.00130 0.0 0 2 3 8 0.00382 0.00615 0.00130 Table 3. The molar depression o f carbon dioxide solubility (Van Slyke e t al., (32). The * denotes t h a t the value is taken f r o m Harned and Davies (12) as the same as t h a t f o r chloride. It lacks d i r e c t experimental support, however. 155 N o r m a l l y f i n a l u r i n e d o e s n o t c o n t a i n s i g n i f i c a n t a m o u n t s of n e i t h e r p r o t e i n s nor lipids. D u r i n g p a t h o l o g i c a l c o n d i t i o n s , h o w e v e r , t h i s could b e t h e case, a n d would t h e n m a k e a s e r i o u s i m p a c t on b i c a r b o n a t e d e t e r m i n a t i o n s . T h e solubility of C O 2 i n c r e a s e s w i t h a h i g h e r lipid c o n c e n t r a t i o n b u t d e c r e a s e s w i t h a high p r o t e i n C o n c e n t r a t i o n . R e n a l e x p e r i m e n t a l t e c h n i q u e s n o w a d a y s p e r m i t s s a m p l i n g f r o m a f f e r e n t a n d e f f e r e n t arterioles as w e l l as f r o m t h e Bowman's capsule. P r i m a r y u r i n e c o n t a i n s p r o t e i n which t h u s will d e c r e a s e t h e b i c a r b o n a t e c o n c e n t r a t i o n . O n t h e o t h e r h a n d o n e c a n e x p e c t a n i n c r e a s e d b i c a r b o n a t e c o n c e n t r a t i o n in p r i m a r y u r i n e as t h e c o n c e n t r a t i o n of t h e p l a s m a p r o t e i n s i n c r e a s e s during t h e u l t r a f i l t r a t i o n . This is d u e t o a n a u g m e n t a t i o n of t h e Donnan effect as w e l l as of a r e d u c e d CO2 s o l u b i l i t y in p l a s m a (Siggaard A n d e r s e n , pp41,(27); S o h t e l l (29)). b) T h e f i r s t a p p a r e n t d i s s o c i a t i o n c o n s t a n t : T h e r e s u l t s f r o m r a t u l t r a f i l t r a t e are shown a t t h e b o t t o m of T a b l e 4. I t m u s t b e born in m i n d t h a t o l d e r l i t e r a t u r e in t h i s f i e l d d e s c r i b e s a c i d - b a s e c h e m i s t r y w i t h a n o t h e r d e f i n i t i o n of pH, t h a n t h a t of t o - d a y (3). F u r t h e r m o r e , t h e a n a l y t i c a l m e t h o d s a r e c o n s i d e r a b l y i m p r o v e d . T a b l e 4 PKa 6.3222 6.105 6.330 6,092 6.089 6.3089 6.09 6.112 6.3002 6.086 6.316 6.103 6.13 6.127 6.328 6.120 6.101 O C 38 38 38 38 38 38 38 37 38 37.5 38 37.5 37.5 37.5 38 37 38 Solution p = o S e r u m ( h u m a n ) H = O S e r u m ( h u m a n ) S e r u m (dog) p = o p = 160 m m o l / l S e r u m (ox+dog+human) p = o p = o S e r u m (dog+human) S e r u m ( h u m a n ) S e r u m (human) p = 160 m m o l / l p = o Amnion fluid U l t r a f i l t r a t e H a s t i n g s and Sendroy, (13) H a s t i n g s et a1.,(14) S t a d i e a n d H a w e s , (31) Robinson et al., ( 2 2 ) -1'- M a c I n n e s and B e l c h e r , (20) Danielson e t al., (6) Dill et al., (7) H a r n e d a n d D a v i e s , (12) S e v e r i n g h a u s et al., (23) F i t z s i m o n s and S e n d r o y , (9) G a m b i n o , (10) -If- (11) Siesjo, (25) S i g g a a r d A n d e r s e n , (26) J o h n e l l , (17) T h i s s t u d y (SE= 0.002, n = 42) T a b l e 4. S u m m a r y of d a t a , d e s c r i b i n g t h e pKa in d i f f e r e n t s o l u t i o n s a t body t e m p e r a t u r e . p= 0 mol/l m e a n s a n e x t r a p o l a t i o n of d a t a f r o m salt solutions. 156 As shown in T a b l e 3, t h e p K a is s t r o n g l y i n f l u e n c e d by t h e ionic s t r e n g t h . A s p e c t r u m of f o r m u l a s i n l i t e r a t u r e are d e s c r i b e d t o s u b s t a n t i a t e t h i s r e l a t i o n . Many f o r m u l a s , b a s e d on t h e Debye-Huckel e q u a t i o n , are r e s t r i c t e d t o t o o d i l u t e d solutions, h o w e v e r , t o b e of i m p o r t a n c e in a n a l y s i s of biological fluids. Fig. 2 v i s u a l i z e s t w o of t h e s e f o r m u l a s , d e s c r i b i n g t h e p K a as a f u n c t i o n of i o n i c s t r e n g t h ( o t h e r r e l a t i o n s are d e s c r i b e d by Manov et al., (21), Hagg, (16), S l a t o p o l s k y e t al. (28) a n d S i g g a a r d A n d e r s e n , (27). T h e f i g u r e a l s o i n d i c a t e s r e a s o n a b l e v a l u e s o f t h e ionic s t r e n g t h in t u b u l a r fluids. I t is c l e a r l y s e e n t h a t t h e w i d e r a n g e in i o n i c s t r e n g t h in t h e s e u r i n e s m a k e s a c o n s i d e r a b l e i m p a c t on t h e p K a a n d t h u s f i n a l l y on t h e b i c a r b o n a t e d e t e r m i n a t i o n . P K h 6.3 6.1 5.9 0.2 0.4 0.6 0.8 1.0 p (MI Fig. 2. p K a as a f u n c t i o n of t h e i o n i c s t r e n g t h (p). I: pKa' = 6.322 - 0.5 c ( H a s t i n g s a n d Sendroy, (13) [I: pKa' = 6.316 - 0.512 f i ( F i t z s i m o n s a n d S e n d r o y , (9)). R e a s o n a b l e m e a n i o n i c s t r e n g t h s f o r p r o x i m a l t u b u l e (PT), e a r l y d i s t a l t u b u l e (DT) and f i n a l u r i n e are i n d i c a t e d . T h e i n f l u e n c e of pH itself on t h e f i r s t a p p a r e n t d i s s o c i a t i o n c o n s t a n t i s s p a r s e l y s t u d i e d in biological solutions. In c e r e b r o s p i n a l fluid (Siesjo, (25)) a n d h u m a n a m n i o t i c fluid ( J o h n e l l , (17)) no i n f l u e n c e of p H on p K a w a s found. In s e r u m , h o w e v e r , as w e l l as in 150 m m o l / l N a C l s o l u t i o n S i g g a r d A n d e r s e n (26) f o u n d a d e c r e a s e in p K a w i t h i n c r e a s i n g pH b u t only a b o v e pH 7.0 - 7.5. S i m i l a r d e p e n d e n c e f o r s e r u m w a s f o u n d by S e v e r i n g h a u s et al. (23). In a l k a l i n e s o l u t i o n s c o n t a i n i n g p r o t e i n s , t h e c a r b a m i n o - C 0 2 c o n c e n t r a t i o n m i g h t i n f l u e n c e on t h e b i c a r b o n a t e a c t i v i t y as i s f u r t h e r discussed by S i e s j o (25). 157 c ) U r i n a r y bicarbonate: Fig. 3 shows a hypothetical urine o f p H 6.8 and Pco2 o f 40 mrn H g (5.3 kPa). Reasonable S and pKa values are furthermore inserted i n the Henderson- Hasselbalch equation and the bicarbonate a c t i v i t y increases w i t h increasing S f o r a certain pKa. The influence o f d i f f e r e n t temperatures on the bicarbonate value i s formulated by Siggaard Andersen (27) and shows that 1% increase, also increases the bicarbonate a c t i v i t y w i t h about 1.6 YO. Thus an analysis o f the bicarbonate concentration i n a solution f r o m a 38OC animal which is analyzed a t room temperature results i n significantly too l o w a value (more than 25%). 8.0 7.0 6.0 5.0 4.0 3.0 610 615 620 6 2 5 6 30 6 35 . . , ~ , . I . 0.0300 0.0315 0.0330 s Fig. 3 The bicarbonate a c t i v i t y as a function o f pKa and S in the Henderson-Hasseibalch equation. The example is based on equilibrium condition w i t h a p H o f 6.8 and a Pco2 of 40 m m Hg. 158 6.0 6.5 7.0 7.5 8.0 pH C 0 2 - e q u i l i b r a t i o n c u r v e s of n o n - b i c a r b o n a t e b u f f e r s . T h e r e a c t i o n e v a l u a t e d i s t h e following: 2- H P O ~ + c 0 2 + H ~ O e H~PO;+ H C O ~ 2- T h e c h a n g e o f H P O 4 --+ H 2 P 0 4 is e x p r e s s e d as A w h i c h also will i l l u s t r a t e t h e e q u i m o l a r i n c r e a s e in HCOj. T h e c u r v e s are c a l c u l a t e d a c c o r d i n g t o S i g g a a r d A n d e r s e n (27). T h e s o l u t i o n s used in t h e c a l c u l a t i o n s are: p h o s p h a t e b u f f e r s w i t h e q u a l c o n c e n t r a t i o n s f o r HzP0; and H P O i - of 2.5 m m o l / l ( l e f t ) a n d 20 mrnol/l ( r i g h t ) a t i o n i c s t r e n g t h s of 75 rnmol/l a n d 300 m m o l / l r e s p e c t i v e l y . In t h e f i g u r e t h e d a s h e d l i n e s i n d i c a t e i s o - b i c a r b o n a t e lines. For c o n c l u s i o n s , see t e x t . T h e e q u i l i b r a t i o n t e c h n i q u e f o r t h e b i c a r b o n a t e d e t e r m i n a t i o n in s o l u t i o n s c o n t a i n i n g n o n - b i c a r b o n a t e b u f f e r s yields e q u i l i b r a t i o n l i n e s which are c u r v e d as shown in Fig. 4. I t is w e l l visualized in t h e f i g u r e t h a t t w o d i f f e r e n t P c o 2 v a l u e s are n o t e n o u g h f o r t h e c o n s t r u c t i o n of t h e e q u i l i b r a t i o n line. 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