Can insulin administration cause an acute metabolic acidosis in vivo?

Diabetologia, Sep 1993

Summary Insulin is the cornerstone of therapy for diabetic ketoacidosis because it causes the rate of ketoacid production to fall; this action takes several hours to occur. Insulin also causes H+ to be transported from the intracellular fluid to the extracellular fluid in vitro. The purpose of this study was to determine if insulin led to the acute export of H+ from the intracellular fluid in vivo. If so, we wished to determine if this also occurred during chronic metabolic acidosis, to quantitate the magnitude of the H+ shift, and to evaluate the mechanisms involved. The administration of low- or high-dose insulin to normal dogs and high-dose insulin to dogs with chronic metabolic acidosis caused the concentration of bicarbonate in plasma to decline by close to 3 mmol/l. The PCO2 fell by close to 15 % in all three groups of dogs, so one component of the fall was due to hyperventilation. As the pH of blood did not change, a primary metabolic acidosis also occurred. The fall in bicarbonataemia was not due to net accumulation of organic acids or to a loss of bicarbonate or organic anions in the urine. Taken together, insulin, when given at doses used to treat diabetic ketoacidosis, might induce a significantly greater degree of acidaemia in the extracellular fluid acutely after it is given.

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Can insulin administration cause an acute metabolic acidosis in vivo?

J. M. Goguen 0 M. L. Halperin 0 0 Division of Nephrology, St Michael's Hospital, University of Toronto , Toronto , Canada C a n i n s u l i n a d m i n i s t r a t i o n c a u s e an a c u t e m e t a b o l i c a c i d o s i s in v i v o ? Summary, Insulin is the cornerstone of therapy for diabetic ketoacidosis because it causes the rate of ketoacid production to fall; this action takes several hours to occur. Insulin also causes H + to be transported from the intracellular fluid to the extracellular fluid in vitro. The purpose of this study was to determine if insulin led to the acute export o f H § from the intracellular fluid in vivo. If so, we wished to determine if this also occurred during chronic metabolic acidosis, to quantitate the magnitude of the H + shift, and to evaluate the mechanisms involved~ The administration of low- or highdose insulin to normal dogs and high-dose insulin to dogs with chronic metabolic acidosis caused the concentration of bicarbonate in plasma to decline by close to 3 retool/1. The Endogenous acid prdduction; intracellular pH; insulin; metabolic acidosis; Na +/H + antiporter; PCO2 9 Springer-Verlag 1993 An experimental study in dogs T h e most c o m m o n association b e t w e e n insulin and m e t a bolic acidosis is the d e v e l o p m e n t of ketoacidosis in which ketoacids are p r o d u c e d due to the combination of high levels of glucagon and a relative lack of insulin [ 1 ]. This ketoacidosis can b e c o m e severe in the patient with Type 1 (insulin-dependent) diabetes mellitus, but is usually mild in degree in chronic fasting [ 2 ]. Insulin m a y influence the degree of acidosis in a n o t h e r way in that it leads to alkalinization of cells in vitro [ 3-5 ]. Thus, administration of insulin might cause acidaemia in the extracellular fluid (ECF) together with a rise in intracellular p H . If H § were t r a n s p o r t e d into the E C F when insulin was administered early in the t r e a t m e n t of a patient with a severe degree of diabetic ketoacidosis ( D K A ) , an acute exacerbation of acidemia might occur. This is particularly critical since during severe metabolic acidosis, most protons are buffered in the intracellular fluid (ICF) [ 6 ]. T h e p u r p o s e of this investigation was to determine if extracellular acidosis occurred in vivo shortly after insulin was administered and if so, to quantitate the extent to which it occurred and provide some insights into its basis. We also addressed the dose of insulin required and if the p h e n o m e n o n was i n d e p e n d e n t of the initial acid-base balance. T h r e e series of experiments were employed: first, a PCO2 fell by close to 15 % in all three groups of dogs, so one component of the fall was due to hyperventilation. As the p H of blood did not change, a primary metabolic acidosis also occurred. The fall in bicarbonataemia was not due to net accumulation of organic acids or to a loss of bicarbonate or organic anions in the urine. Taken together, insulin, when given at doses used to treat diabetic ketoacidosis, might induce a significantly greater degree of acidaemia in the extracellular fluid acutely after it is given. n o r m a l acid-base series given a high dose of insulin (a dose similar to that used in vitro [ 5 ] ), second, a normal acidbase series given a lower dose of insulin (one that is used to treat D K A ) and third, a series with chronic metabolic acidosis given the high dose of insulin. Results to be rep o r t e d indicate that m o r e severe acidosis developed in the E C F in all three series of experiments. T h e underlying m e c h a n i s m for the E C F acidosis was also e x a m i n e d by quantitating e n d o g e n o u s net acid production, the shift of cations across the ECF: I C F interface, the loss of bicarbonate and changes in the PCO2. Materials and methods Chemicals: Enzymes, cofactors and metabolic intermediates were obtained from Sigma Chemical Company (St. Louis, Mo., USA). All other reagents were of the highest purity available. Animals: Mongrel dogs of either sex (19 _+1 kg) were prepared in two ways: dogs with normal acid-base balance were fed their usual diet, and dogs with chronic metabolic acidosis received 5 mmol NH4C1/kgbody weight twice a day with meals for 5 days. Food was withheld on the morning of study. Experimentalprocedure: Dogs were anaesthetized with phenobarbital (25 mg/kg i. v. with additional doses as needed to maintain a con Blood pH PCO2 HCO3 Urine HCO3NH4 + (mmHg) (mmol/1) (mmol/1) (mmol/1) (mmol/1) (retool/l) (meq/1) (g/l) (gmol/min) (gmol/min) ap < 0.05 for paired values. For details, see Methods. Results from the 60-rain control period and the 40 to 60-rain period after insulin administration (mean + SEM) are shown. Urine was collected for the entire 60 min of the insulin stant depth of anaesthesia throughout the experiment). The dogs were permitted to breathe spontaneously throughout the experiment. The abdomen was opened by midline incision and catheters were inserted into both ureters, the internal jugular veins for infusions and in the femoral artery for blood sampling. To maintain a normal ECF volume and urine output after surgery, each dog received a continuous i.v. infusion containing 140 mmol/1 NaC1, 10 mmol/1 KC1 and 82 mmol/1 mannitol at a rate of 6.7 ml/min for 60 rain after the surgery; at this point the infusion rate was reduced so that the total input (1.4 ml/min) matched the rate of urine output. Urine was collected for 15-min periods and an arterial blood sample was obtained at the beginning and end of each period. The values in plasma in this 60-min control period did not vary appreciably and represent the 'before insulin' value. At the end of the control period, five normal dogs received a bolus of 0.1 IU/kg regular beef and pork insulin and this was followed by a constant infusion of 0.1 IU. kg l.h 1 Nine normal dogs and 14 dogs with metabolic acidosis received the high-dose insulin protocol - a bolus of 1.5 IU/kg body weight followed by a constant infusion of 1.8 IU insulin, kg- 1.h - 1 Samples of blood were obtained at 20, 40 and 60 min and urine was collected as described above for the three 20-min collections after the bolus of insulin was administered; a steady state was achieved by 40 min. Six additional normal dogs and five additional dogs with metabolic acidosis did not receive insulin and had the control period extended for an additional i h to serve as time controls. Analytical methods: The pH, PCO2 and PO2 of blood and the pH of urine were measured anaerobically at 37 ~ with a digital acid-base analyser (Coming 178 blood pH analyzer, Medfield, Mass., USA). Analytical methods for haemoglobin, metabolites, oxygen, sodium, potassium and creatinine were as previously described [ 7 ]. The concentration of bicarbonate in plasma was calculated using a pK' of 6.10 and a solubility factor for CO2 of 0.0301 [ 8, 9 ]. Insulin was measured by radioimmunoassay. Statistical analysis Results are reported as the mean + SEM. For each dog, the values at the end of the control period were compared to values (at 40 and 60 min) in the insulin period; since the latter two values were vir Normal dogs low-dose insulin (n = 5) Before period. Urinary net charge is a marker of urinary anion excretion and is defined as the urinary (Na++ K + - C 1 ) x flow. CMA, chronic metabolic acidosis tually identical, they were treated as single values for ease of reporting. Statistical comparisons were performed by paired analyses on data from individual dogs using the two-tailed Student's t-test. R e s u l t s A l l t h e d o g s h a d t h e e x p e c t e d fall in t h e c o n c e n t r a t i o n s o f p l a s m a g l u c o s e , K + a n d p h o s p h a t e w i t h i n 40 m i n a f t e r insulin was a d m i n i s t e r e d ( T a b l e 1). I n e a c h s e r i e s o f e x p e r i m e n t s , t h e r e w a s also a s i g n i f i c a n t fall in t h e c o n c e n t r a t i o n o f b i c a r b o n a t e in p l a s m a ; this fall w a s 4.7 + 0.6 mmol/1 in t h e n o r m a l d o g s g i v e n a h i g h d o s e o f i n s u l i n a n d 2.5 + 0.7 mmol/1 in t h e n o r m a l d o g s g i v e n t h e l o w e r d o s e o f i n s u l i n ( T a b l e 1). I n t h e a c i d o t i c dogs, t h e fall in p l a s m a b i c a r b o n a t e w a s 3.2 + 0.5 mmol/1, t h u s t h e r e w a s a l a r g e r p r o p o r t i o n a t e fall in t h e c o n c e n t r a t i o n of b i c a r b o n a t e in p l a s m a in t h e s e dogs. I n six a d d i t i o n a l n o r m a l d o g s a n d five d o g s w i t h c h r o n i c m e t a b o l i c a c i d o s i s t h a t d i d n o t r e c e i v e insulin, t h e r e w e r e n o s i g n i f i c a n t c h a n g e s in p l a s m a p H , b i c a r b o n a t e , l a c t a t e o r t h e a n i o n g a p in a s e c o n d 60m i n t i m e c o n t r o l p e r i o d ( r e s u l t s n o t s h o w n ) . To p r o v i d e insights i n t o t h e u n d e r l y i n g m e c h a n i s m o f t h e E C F a c i d o s i s i n d u c e d b y insulin, s e v e r a l p o s s i b i l i t i e s w e r e e x a m i n e d . F i r s t , t h e r e w a s n o e v i d e n c e o f o n g o i n g e n d o g e n o u s n e t a c i d p r o d u c t i o n in a n y d o g as t h e a n i o n g a p in p l a s m a d i d n o t rise a p p r e c i a b l y n o r d i d t h e u r i n e c o n t a i n a n i n c r e a s e in u n m e a s u r e d a n i o n s f o l l o w i n g insulin a d m i n i s t r a t i o n ( T a b l e 1). F u r t h e r , a l t h o u g h t h e r e w a s a s m a l l rise in t h e c o n c e n t r a t i o n o f L - l a c t a t e in b l o o d , this r i s e was less t h a n 1 mmol/1 f o l l o w i n g t h e a d m i n i s t r a t i o n o f i n s u l i n ( T a b l e 1). S e c o n d , t h e r e w a s n o e v i d e n c e o f loss o f a n a p p r e c i a b l e q u a n t i t y o f b i c a r b o n a t e in t h e u r i n e , b u t g a s t r o i n t e s t i n a l e x c r e t i o n s w e r e n o t e x a m i n e d . T h i r d , a fall in b l o o d P C O 2 c a n r e s u l t in a r e d u c t i o n in t h e c o n c e n t r a t i o n o f b i c a r b o n a t e d u e to t h e shift o f H + o u t o f cells down their concentration gradient; in the data reported in Table 1, the fall in PCO2 was not associated with a rise in blood pH. Thus, the data were most consistent with the shift of H + ions out of cells, due in part to a fall in PCO2 and not due to endogenous net acid production. A n y shift of H + ions out of cells must be accompanied by a shift of the equivalent amount of cation into cells or of an anion out of ceils to maintain electroneutrality. T h e fall in the concentration of K § in plasma was much less than the fall in bicarbonate. Also, there was a net shift of phosphate into cells (Table 1). Thus, if there was an exit of H § from cells, it was probably accompanied by the entry of Na + to maintain electroneutrality. Discussion The purpose of this study was to determine if acute administration of insulin would cause a shift of H + into the E C F as suggested by in vitro experiments [ 3, 4, 5, 10 ]. In all three dog models, the actions of insulin were evident because there was a fall in the concentrations of glucose, K + and phosphate in plasma. A major new observation was the significant fall in bicarbonate concentration in plasma shortly after insulin acted. During the net production of acids, a neutral comp o u n d such as glucose is converted to an anion such as lactate (plus a H +) [ 11 ]. This anion must be retained in the body or excreted without H + or NH4 + to produce a net gain of H + in the body [ 12 ]. Therefore, the hallmark of net endogenous acid production is the accumulation of new anions in the body or urine [ 13 ]. In all three models, the anion gap in plasma (Na + + K § - C1- - HCO3 ) did not rise during the insulin period, nor was there a net excretion of anions without H + or NH4 + ions, Also, the concentration of L-lactate in blood rose by only a fraction of a mmol/1. Thus, it is unlikely that endogenous net acid production accounted for the fall in bicarbonate concentration. The content of bicarbonate in the E C F could fall if sodium bicarbonate was lost from the body either in the urine or gastrointestinal secretions. In all three models, there was no significant loss of bicarbonate in the urine. The volume and concentration of bicarbonate was not measured in the gastrointestinal secretions, so this must remain a possible explanation. Nevertheless, there are no data reported to suggest that insulin causes metabolic acidosis due to a t e m p o r a r y loss of bicarbonate via the gastrointestinal tract. If anything, insulin may augment the secretion of HC1 in the stomach and raise the concentration of bicarbonate in the E C F [ 14 ]. If bicarbonate disappeared due to conversion to carbon dioxide and water, the source of H* was probably the ICF as there are too few H § (free or buffered) in the E C F to consume a large quantity of bicarbonate. T h e shift of H + could occur either due to a fall in the PCO2, or due to a more direct effect of insulin. Considering a fall in PCOa first, an acute fall should lead to a rise in blood p H if this were the sole cause for the observed fall in the concentration of bicarbonate - there was no rise in p H of plasma during the insulin period. H e n c e there must be an additional mechanism to explain this fall in bicarbonate. In quantitative terms, the expected fail in bicarbonate when dogs were hyperventilated is close to 2.5 mmol/1 for each halving of the PaCO2 [ 15 ]. The final possible explanation for the fall in bicarbonate concentration in plasma to consider is that insulin led to a shift of H + out of cells independent of respiratory alkalosis. This m o v e m e n t can only occur if a cation enters the ICF in exchange for H +, or if an anion leaves the ICE Since there were no new anions found in the ECF, a cation (Na § and/or K +) should have entered the ICF to maintain electroneutrality. Since the decline in bicarbonate concentration exceeded the fall in K § in plasma, by process of elimination, the most likely cation present in sufficient quantity in the E C F for this shift with H + is Na +. Unfortunately, balance studies are not accurate enough to detect a 16 mmol loss of Na + (4 mmol/1 x the E F C volume of 4 litres) in a total pool of Na § in the E C F of close to 600 mmol. T h e r e are data obtained in vitro to support the hypothesis that insulin induces a rise in intracellular p H due to the export of H + - examples include myocytes [ 3, 5 ], renal proximal cells [16] and in some studies in adipocytes [ 17, 18 ]. When insulin was added to muscle cells in vitro, there was a rapid, significant and sustained rise in intracellular p H which could be abolished by replacing Na § in the medium with another cation or by adding amiloride in a quantity sufficient to block the Na +/H + antiporter [ 4, 5 ]. The lower the initial ICF pH, the greater was the rise in ICF p H induced by insulin [ 19 ]. To determine if the effect of insulin on ICF and E C F p H could occur in a physiological setting, the concentration of insulin required to elicit this change needs to be examined. In experiments with myocytes, the effect on intracellular p H required very high concentrations of insulin ranging from 2 10 -8 mmol/1 to 1 x 10 -6 mmol/1 (ICF p H changed from 0.03 to 0.14 units, respectively). Dogs receiving high-dose insulin had measured circulating insulin levels close to 2 x 10 -7 mmol/1. It might be important from a clinical perspective to consider the impact of a shift of H + following the administration of insulin. Insulin is administered to suppress the production of ketoacids during therapy for D K A , an action that requires several hours. If the Na +/H § antiporter was activated acutely, the result would be both a higher amount of H § in the E C F and a higher amount of Na § in the ICF, both of which could conceivably be detrimental; possibly, the rise in intracellular p H might be beneficial. A second instance where activation of the Na/H + ion antiporter could be important focuses on a rise in intracellular Na § concentration. F o r example, Na + is an effective osmole, unlike the H + it replaces (the latter did not contribute to osmolality when it was bound to proteins); thus water should enter cells and result in cell swelling. This possibility has b e e n raised as an explanation for the cerebral o e d e m a that can occur following treatment with insulin [ 20 ]. T h e r e are other possible ways that a rise in intracellular Na + can influence body function. In the patient with essential hypertension, hyperinsulinaemia could exacerbate the degree of hypertension if it stimulates the N a + / H § pump in smooth muscle cells. A n increased [Na § in their I C F could lead to an increase in intracellular [Ca 2+] and thus to s m o o t h muscle contraction a n d i n c r e a s e d p e r i p h e r a l resistance. Finally, the m e t a b o l i c alkalosis which follows glucose feeding to obese, chronically fasted subjects m a y be a n o t h e r possible m a n i f e s t a t i o n of stimulation of the N a § + antip o r t e r by insulin [ 21 ]. Should insulin stimulate the N a § + a n t i p o r t e r in the luminal m e m b r a n e of cells of the p r o x i m a l c o n v o l u t e d tubule, this w o u l d result in N a + r e a b s o r p t i o n and H § secretion. A s a result, the increased m a x i m a l r e a b s o r p t i v e capacity for b i c a r b o n a t e and the higher renal b i c a r b o n a t e threshold t h a t o c c u r r e d w h e n these patients w e r e refed could b o t h be explained by this mechanism. I n summary, in t h r e e different settings, an E C F acidosis resulted following the administration of insulin in vivo. This effect was dose d e p e n d e n t and o c c u r r e d in b o t h normal dogs and those with chronic m e t a b o l i c acidosis. O u r data are m o s t consistent with this effect being due to a fall in PCO2 a n d the export of H + f r o m cells. N u m e r o u s in vitro m o d e l s s h o w that insulin can stimulate the N a + / H § a n t i p o r t e r and result in intracellular alkalosis. F u r t h e r studies are r e q u i r e d to d e t e r m i n e if this effect also occurs in patients with D K A after treatmer/t with insulin. Acknowledgements.The authors are very grateful to Dr. S. Cheema Dhadli and K. S. Kamel for helpful discussions and critique during the preparation of the manuscript. We are also indebted to Mr. C. Bun-Chen, Ms. S. Tang and Ms. E. Singer for their technical assistance and to Ms. J. Mangat for secretarial assistance. 1. McGarry JD , Woeltje KF , Kuwajima M , Foster DW ( 1989 ) Regulation of ketogenesis and the renaissance of carnitine palmitoyltransferase . Diab Metab Rev 5 : 271 - 284 2. 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J. M. Goguen, M. L. Halperin. Can insulin administration cause an acute metabolic acidosis in vivo?, Diabetologia, 1993, 813-816, DOI: 10.1007/BF00400355