The lactate-to-pyruvate reaction for serum lactate dehydrogenase (LD) is most frequently assayed in one of three buffers, pyrophosphate (PPi), tris(hydroxymethyl)amino-methane (Tris), or 2-amino-2-methyl-1-propanol (AMP). We described interconverting results for serum samples and for highly purified LD isoenzymes I (dissolved in one of these matrixes) assayed in these buffers under optimized reaction conditions. The equation for converting results obtained for sera in Tris (x) to those in PPi(y) (both at 30 degrees C) is y = 0.74x+10 (n = 98). Since AMP is used extensively in Technicon procedures, we determined the LD activity of sera with an SMA 12/60, at 37 degrees C. The equation for convering these AMP results to results obtained in PPi at 30 degrees C is y = 0.45x-16 (n = 90). Very different equations were obtained with highly purified LD isoenzyme I maintained in two different matrixes and with both isoenzymes assayed in the same matrix. The matrix in which LD is dissolved and the proportion of various LD isoenzymes affect the magnitude of difference in observed LD activity under various conditions. Therefore, in clinical laboratories that use more than one analytical method or when conversion equations are used in the comparison of interlaboratory results, it is important to define the LD source, isoenzyme content, and the matrix, as well as the reaction conditions, and to use many samples with a wide range of activities when determining the conversion equations. For any changes in reagent source, substrate concentration, or alteration in procedure, a new normal range and new conversion equations should be determined.

Optimal reaction conditions to sassay human lactate dehydrogenase (lactate-to-pyruvate) were established for isoenzymes 1 and 5 at 25, 30, and 37 degrees C in diethanolamine and 2-amino-2-methyl-1,3-propanediol. Different substrate concentrations are required at each temperature. The conditions permit measurement of lactate dehydrogenase 1 and 5 with the lowest substrate concentrations that allow for the highest equal sustainable efficiency in measuring both isoenzymes. About 95% of each isoenzyme activity is measured if the assay is performed within the first minute after the reaction is initiated even for activities as high as triple the upper limit of normal. The Arrhenius relationship is different for each isoenzyme, but results obtained for each at one temperature can be compared with results at another temperature by use of simple conversion equations. Assays at 25 and 30 degrees C are more economical and less variable than assays at 37 degrees C.

Human lactate dehydrogenase isoenzymes I and V have decreased activities when the reaction is initiated with lactate. No loss in lactate dehydrogenase I activity was found when the reaction was initiated with enzyme or NAD+. For lactate dehydrogenase V an NAD+-initiated reaction, as compared to an enzyme-initiated reaction, yields lower activity in sodium pyrophosphate buffer but higher activity in tris(hydroxymethyl)aminomethane buffer. Both isoenzymes have higher lactate-to-pyruvate activity when assayed in the latter buffer than when assayed in the former. Human lactate dehydrogenase V (but not I) exhibited different activities when assayed with lactate from two different commercial sources. Human lactate dehydrogenase assayed by the pyruvate-to-lactate reaction is not affected by the choice of reaction initiator.

Hydrogen ion secretion by the kidneys of anesthetized hypothermic dogs was measured to ascertain if the hypothermic kidney helps to compensate for the acidosis which develops during exposure to cold. Artificial respiration was not used. Total hydrogen ion excretion was measured as the sum of total titratable acid and ammonia excreted. A constant saline infusion of 2 ml/min was employed to maintain a urine flow adequate for analysis procedures. Though anaerobic arterial pH, corrected to body temperature, fell significantly from 7.41 during control to 7.27 at 27 °C, urinary pH remained at control levels of approximately 6.8. Neither titratable acid nor ammonia excretion increased in response to the hypothermic acidosis as would have occurred during normothermia with a similar low pH. Total titratable acid excretion fell from a control value of 0.011 meq/min to 0.005 meq/min at 27 °C. Ammonia excretion, after a mild increase from a control value of 0.008 meq/min, fell to 0.006 meq/min at 27 °C. The failure of the hypothermic kidney to excrete additional hydrogen ions in the face of acidosis indicates that the renal acidification mechanism is temperature-dependent.

The investigation was designed to determine whether the renal loss of bicarbonate contributes significantly to the acidosis of hypothermia. The excretion of bicarbonate during hypothermia was studied in five infused (6% creatinine in saline at 0.4 ml/minute) and five non-infused dogs. All animals were anesthetized and artificial respiration was not used. The rectal temperature was gradually reduced to the 26–27 °C range by approximately 4 hours of exposure to ice packing. After control, clearance periods of 30 minutes' duration were conducted serially and continually through the experiment. There was no significant increase in bicarbonate excretion during hypothermia in the non-infused group. The urinary pH remained at control levels of about 6.2. The fall in arterial pH was not due to urinary bicarbonate loss. The urinary pH in the infused group, which had a higher urine flow, increased to pH 6.7 due to increased excretion of bicarbonate. The urine pH in three animals with highest urine flows in this latter group approached plasma levels. The excretion rate of bicarbonate in the infused group, however, was similarly insufficient to account for the decrease in arterial pH. The hypothermic kidney is quite effective in reabsorbing bicarbonate.