13CO2 recovery fraction in expired air of septic patients under mechanical ventilation

Braz J Med Biol Res, July 2008, Volume 41(7) 563-570

¹³CO₂ recovery fraction in expired air of septic patients under mechanical ventilation

M.A. Martins¹, F.A. Coletto¹, O.A. Martins-Filho³, J.S. Marchini² and A. Basile-Filho¹

¹Centro de Terapia Intensiva (Campus), Hospital das Clínicas, Departamento de Cirurgia e Anatomia, ²Laboratório de Espectrometria de Massa, Departamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil

³Laboratório de Biomarcadores de Diagnóstico e Monitoração, Instituto René Rachou, Fundação Oswaldo Cruz, Belo Horizonte, MG, Brasil

References ^{Correspondence and Footnotes}

Abstract

The continuous intravenous administration of isotopic bicarbonate (NaH¹³CO₂) has been used for the determination of the retention of the ¹³CO₂ fraction or the ¹³CO₂ recovered in expired air. This determination is important for the calculation of substrate oxidation. The aim of the present study was to evaluate, in critically ill patients with sepsis under mechanical ventilation, the ¹³CO₂ recovery fraction in expired air after continuous intravenous infusion of NaH¹³CO₂ (3.8 µmol/kg diluted in 0.9% saline in ddH₂O). A prospective study was conducted on 10 patients with septic shock between the second and fifth day of sepsis evolution (APACHE II, 25.9 ± 7.4). Initially, baseline CO₂ was collected and indirect calorimetry was also performed. A primer of 5 mL NaH¹³CO₂ was administered followed by continuous infusion of 5 mL/h for 6 h. Six CO₂ production (VCO₂) measurements (30 min each) were made with a portable metabolic cart connected to a respirator and hourly samples of expired air were obtained using a 750-mL gas collecting bag attached to the outlet of the respirator. ¹³CO₂ enrichment in expired air was determined with a mass spectrometer. The patients presented a mean value of VCO₂ of 182 ± 52 mL/min during the steady-state phase. The mean recovery fraction was 0.68 ± 0.06%, which is less than that reported in the literature (0.82 ± 0.03%). This suggests that the ¹³CO₂ recovery fraction in septic patients following enteral feeding is incomplete, indicating retention of ¹³CO₂ in the organism. The severity of septic shock in terms of the prognostic index APACHE II and the sepsis score was not associated with the ¹³CO₂ recovery fraction in expired air.

Key words: Recovery fraction; ¹³CO₂; Stable isotope; Septic shock; Intensive care setting

Introduction

Sepsis, septic shock and multiple organ dysfunctions are the main causes of death in intensive care units despite the recent great technological and scientific advances and efforts devoted to improving outcome (1-3).

Several aspects deserve special attention regarding septic patients. In addition to hemodynamic, infectious and immune response evaluations, metabolic parameters are also relevant. Organic metabolism is a dynamic process, especially in pathological situations, with protein synthesis and degradation continuing to occur. Carbon dioxide (CO₂) is produced mostly due to the oxidation of substrates, which can be quantified by using stable isotopes. The endocrine-metabolic response triggered by sepsis cannot be avoided but can be treated by preventing greater tissue injury on the basis of a detailed knowledge of metabolism. One of the most important metabolic changes occurring during sepsis/septic shock is the predominance of the catabolic response of skeletal muscle. This response is multifactorial and reflects the inhibition of amino acid uptake and the reduction of protein synthesis, with accelerated protein breakdown (4). Protein degradation exceeds protein synthesis leading to increased O₂ consumption (VO₂), which, if not corrected, can be associated with multiple organ dysfunctions.

The measurement of nutrient oxidation rate in critically ill patients receiving nutritional therapy is of great interest (5-7). There are few reports using stable isotopes such as labeled carbon (¹³C) in sepsis/septic shock. It is possible to obtain the CO₂ recovery fraction in expired air by administering labeled bicarbonate intravenously (NaH¹³CO₂) (8). Since the CO₂ produced from the labeled substrate interacts with the bicarbonate pool before being expired, it is necessary to determine this ¹³CO₂ recovery fraction in expired air in order to study the oxidation rate for a given substrate (for example, protein). This recovery fraction can be used as a correction factor in the calculation of protein oxidation in subsequent studies.

The objective of the present study was to measure the ¹³CO₂ recovery fraction in expired air of critically ill patients with sepsis or septic shock on mechanical ventilation who were receiving enteral or parenteral nutritional treatment, by continuous intravenous infusion of NaH¹³CO₂.

Subjects and Methods

Subjects

A prospective clinical study was conducted in the Intensive Care Center (Campus) of the University Hospital, Faculty of Medicine of Ribeirão Preto. The study was approved by the Research Ethics Committee of the University Hospital, Faculty of Medicine of Ribeirão Preto and written informed consent was obtained from the patients or persons responsible for them (Process #4989/99, HCRP). The investigation involved 10 patients (4 men and 6 women aged 15 to 85 years with sepsis of any origin or septic shock after blood volume restoration and hemodynamic stabilization. We included patients in the period between the 2nd and 5th day of evolution of the clinical picture, with an indication for invasive mechanical ventilation, with oxygen fraction in inspired air (FiO₂) <0.6, mean arterial pressure >50 mmHg, hourly diuresis >50 mL/h, and requiring the use of nutritional therapy. Exclusion criteria were: oliguric renal insufficiency of any etiology, spontaneous ventilation, brain death and refusal to participate by the patient or person responsible. The criteria used for the diagnosis of sepsis and septic shock were those established by the consensus conference of the American College of Chest Physicians/Society of Critical Care Medicine (9). The patients were divided into two groups according to severity as determined by the prognostic index APACHE II (Acute Physiologic and Chronic Health Evaluation) (10) and by sepsis score (11). All patients were submitted to blood volume restoration, mechanical ventilatory support with a microprocessor-controlled ventilator (Bird 8400 STi Bird Prod Corp., USA; Servo 900C Siemens, Sweden; Savina Draegger, Germany), appropriate antibiotic therapy, and the use of vasoactive drugs and nutritional therapy according to the real energy expenditure calculated by indirect calorimetry (70% carbohydrates, 30% lipids in relation to total calorie value, and approximately 1 g·kg^-1·day^-1 protein) according to the diet standardized by HCFMRP-USP. The patients were sedated with benzodiazepines and/or opiates, and a muscle blocker was used when necessary. Indirect calorimetry was performed on all patients using a portable Deltatrac II Metabolic Monitor (Datex-Ohmeda, Finland) coupled to the mechanical ventilator. Barometric and gas pressure were calibrated before each set of measurements. Calorimetry was performed during the 6 h of the protocol, and mean VCO₂ was calculated for each hour together with expired air collection. The patients were weighed using a portable scale (Slingscale 2002, Instrucom/Hill-Rom series, Hillenbrand Industries, USA).

Patient age ranged from 28-78 years (median 56 years; mean 55.1 ± 19 years). The APACHE II prognostic index ranged from 17-37 (median 24.5; mean 25.9 ± 7.4), with a calculated death risk with a median of 45 (29-88) and a mean value of 60 ± 20%. Sepsis score showed a median value of 18 and ranged from 11-26 with a mean of 19.1 ± 4.2. The intravenous use of the stable isotope (NaH¹³CO₂) did not alter the acid-base status of the patients, as confirmed by arterial gas measurement before and after the study. Of the 10 patients studied, 8 (80%) died within 5 days.

Experimental design

The protocol lasted 6 h. Basal expired CO₂ was determined before starting the infusion of bicarbonate with labeled carbon (3.8 µmol/kg NaH¹³CO₂ diluted in 0.9% NaCl in ddH₂O) and indirect calorimetry was used throughout the study. After basal gas collection, 5 mL NaH¹³CO₂ was infused in bolus, followed by continuous infusion at 5 mL/h for 6 h (T1 to T6). The mean value obtained for each hour (time) of study was used as the basis for the calculation of ¹³CO₂ recovery fraction in expired air. For the calculation of the recovery fraction, we used VCO₂ (converted from mL/min to mmol·kg^-1·h^-1) obtained by indirect calorimetry and the ¹³CO₂ value in expired air.

The dose of NaH¹³CO₂ was calculated according to the weight of the patient on the day when the protocol was applied. The dose used for bolus infusion (prime) was 3.8 µmol/kg and the dose for continuous infusion was 3.8 µmol·kg^-1·h^-1, based on the study of Tissot et al. (12). The stable isotope used was NaH¹³CO₂ (99 atm%; Mass Trace Technologies, USA). The isotope was prepared in the Pharmacy Division of the University Hospital of Ribeirão Preto under a laminar flow hood and under aseptic and antiseptic conditions.

Arterial gases were measured before and after the study to determine possible interferences of the amount of NaH¹³CO₂ with the metabolic state of the patient.

Expired air was initially collected to establish a basal reference value at times -40, -25, and -15 min before the beginning of isotope infusion. Two samples per hour were then collected throughout the procedure (6 h of isotope infusion). Collection was performed with a 750-mL gas collecting bag (Quintron, USA) connected to the expiratory outlet of the mechanical ventilator. The samples were analyzed by mass spectrometry using isotope ratio mass spectrometry (Europa Scientific, England).

Equations used for the calculation of VCO₂, the ¹³CO₂ enrichment as well as ¹³CO₂ recovery fraction in expired air samples were previously described in the literature (13,14). The value for Enr ¹³CO₂ APE (enrichment of expired air in percent of excess atoms) x 1000 refers to the arithmetic difference between the ¹³CO₂ values obtained for expired air samples and the basal values before NaH¹³CO₃ administration.

Statistical analysis

Data sets were first evaluated by Minitab 13.20 software (USA) to test three hypotheses: independence, normality and variance in order to assess their parametric or non-parametric nature. Considering the non-parametric nature of all data sets, comparison between the two groups studied according to severity was performed using the non-parametric Wilcoxon sign post-test for two correlated samples, with the level of significance set at P < 0.05. Data are reported as median (min-max) as well as mean ± SD. Additionally, non-parametric Spearman correlation coefficients were also determined between ¹³CO₂ recovery fraction and the minute volume and the VCO₂.

Results

The median energy expenditure for the patients was calculated to be 1493 ranging from 1131-2353 and mean value of 1587 ± 430 kcal/min, with a value of 1440 ± 121 kcal/min when the Harris-Benedict equation (14) without correction factors was used, avoiding the super-estimation that the correction factor yields in the energy expenditure values. Using this approach, the difference between the two methods (146 kcal/min) was non-significant (P = 0.43). The median value of minute volume was 7.6 (5.2-11.1) with a mean of 7.7 ± 1.8 L/min, indicating that the alveolar minute ventilation was within normal limits. This suggested a low possibility of interference of CO₂ elimination with the recovery fraction since partial CO₂ pressure (paCO₂) remained stable. Mean CO₂ production was 182 ± 52 mL/min, also corresponding to values within normal limits (Table 1).

Median O₂ consumption was 216 (140-343) with a mean of 229 ± 67 mL/min, which remained stable throughout the study. This was expected because the patients, despite their septic shock condition, were stable due to the hemodynamic support provided. The median respiratory quotients of 0.8 ranging from 0.57-0.90 with a mean of 0.79 ± 0.10 characterized an adequate metabolic state, with no predominance of lipolysis, of gluconeogenesis or of lipogenesis (Table 1).

Mean ¹³CO₂ enrichment in expired air was 4.01 ± 0.76 (APE x 1000) during the period from 120 to 360 min. Dynamic evaluation throughout the study demonstrated the presence of a steady state at about 2 h after the beginning of isotope infusion, an expected result due to the administration of the initial priming dose. A gradual increase in enrichment occurred thereafter, reaching a steady state in 120 min. No significant difference in enrichment was observed between groups of different septic severity (P = 0.99) (data not shown).

There was a positive, although weak, correlation between enrichment and minute volume and real energy expenditure, suggesting that alveolar ventilation and the diet offered somehow affected the percentage of ¹³CO₂ enrichment in expired air.

The median ¹³CO₂ recovery fraction in expired air was 0.70 (range 0.55-0.74) with a mean of 0.68 ± 0.06, differing from those reported for non-septic critically ill patients, which suggests that at some time during the course of septic shock there may have been a shift of CO₂ to other metabolic pathways. Comparison of the ¹³CO₂ recovery fraction between different groups according to age (P = 0.81), sex (P = 0.25) and severity of disease (P = 0.62) did not identify statistically significant differences (data not shown). Again, comparison of patient groups selected according to serum urea and bicarbonate levels did not reveal statistically significant differences (P = 0.99 and P = 0.25, respectively) (data not shown).

When groups were compared according to VCO₂ values, we observed that 2 patients had VCO₂ above normal (>200 mL/min) and 8 patients had VCO₂ <200 mL/min. When groups categorized according to VCO₂ values and APACHE II above and below the mean were compared, 7 patients were found to present APACHE II above the mean and 3 were found to present APACHE II below the mean. Among the 7 patients with APACHE II above the mean, 5 presented VCO₂ above the mean, and all 3 patients with APACHE II below the mean presented VCO₂ below the mean.

Regarding the route used for nutritional therapy, no significant difference was observed between the 3 patients receiving parenteral nutrition and the 7 patients receiving enteral nutrition (P = 0.5) (data not shown).

There was a positive correlation between recovery fraction and real energy expenditure (r = 0.62; Figure 1). There was also a positive, although weak, correlation between recovery fraction and minute volume (r = 0.58) as well as VCO₂ and VO₂ (r = 0.75, r = 0.57; Figure 2).

Figure 1.
Correlation of the ¹³CO₂ recovery fraction in expired air and the real energy expenditure for the 10 patients studied. Spearman correlation test was used for statistical analysis as described in Methods (P < 0.05).

Figure 2.
Correlation of the ¹³CO₂ recovery fraction in expired air and the minute volume, VCO₂ and VO₂ values for the 10 patients studied. Spearman correlation test was used for statistical analysis as described in Methods (P < 0.05).

Table 1.
Real energy expenditure, minute volume, CO₂ production (VCO₂), oxygen consumption (VO₂), respiratory quotient, and ¹³CO₂ recovery fraction in the patients studied.

Table 2.
Measurement of ¹³CO₂ recovery fraction in expired air during NaH¹³CO₂ infusion in different situations.

Discussion

In the present study, we evaluated for the first time the ¹³CO₂ recovery fraction in expired air from septic patients divided into groups according to severity. One of the initial objectives was also to correlate the ¹³CO₂ recovery fraction in expired air with the metabolic variable obtained by indirect calorimetry, i.e., VO₂, VCO₂, minute volume, and real energy expenditure.

The use of ¹³CO₂ permits the study of CO₂ metabolism and of its pathways of action. The technique is based on the principle of injection of a substance enriched with ¹³C into blood circulation and evaluation of the recovery fraction of this substance in collected expired air. Furthermore, the ¹³CO₂ estimation is required for whole-body protein turnover, using the ¹³C-leucine technique (13). Indeed, protein kinetics can be quantified from the rate at which labeled leucine is released from protein and the amount of leucine per gram protein. The leucine oxidation is calculated from the ratio of its first step metabolite α-ketoisocaproic acid (¹³C-KIC measured in blood) and ¹³CO₂ production, corrected for the retention of ¹³CO₂ in the bicarbonate pool (fractional recovery). Moreover, several studies have assessed the ¹³CO₂ recovery fraction in experimental animals and in humans by infusing NaH¹³CO₂, with results ranging from 0.51 to 0.95 (Table 2) (8,12,15-22).

Several studies have reported variation in the ¹³CO₂ recovery fraction in adults (17,20,22,23). In resting adults, this rate may range from 0.52 to 0.94 during fasting and from 0.72 to 0.90 during feeding. Several factors may explain the discrepancies detected in these studies, such as patient age, metabolic or nutritional status, the presence of underlying pathological conditions, and, especially, the conditions under which the experimental protocol was carried out.

The few studies available about critically ill patients under mechanical ventilation did not distinguish between specific diseases and this cannot be extrapolated to any one specific disease. According to Tissot et al. (12), ¹³CO₂ recovery fraction in expired air of stable critically ill patients under mechanical ventilation was 0.89 ± 0.26, suggesting that a percentage is retained in the organism, with possible utilization in other metabolic pathways that have not been fully clarified.

The principle behind the use of stable isotopes assumes that the element (carbon 13) will be recognized by the organism as the natural element which already exists, since the two elements share the same chemical properties. The isotope will have to reach equilibrium within pre-existing metabolic pathways. The quantity of the isotope that enters the pool is equal to the amount that leaves the pool. In this case, the entry is represented by the administration of the substance and the exit by its oxidation. In order to reach stability more rapidly, in the present study a priming dose of the isotope that led to steady state within approximately 120 min was administered. Without this priming dose, this equilibrium would have been reached within an interval as long as 24 h (8). The duration of the present study was 360 min in order to provide a safe and detailed evaluation of the kinetics of ¹³CO₂. When NaH¹³CO₂ was administered intravenously and its recovery fraction in expired air was determined, we noted that the isotope was not fully recovered. Clinical studies evaluating the ¹³CO₂ recovery fraction in expired air have reported values ranging from 0.52 to 0.95. Values below 1 indicate that the injected isotope fraction is not fully eliminated as ¹³CO₂ in expired air. Three main destinations have been identified thus far for the retention of this isotope in the organism. First, ¹³CO₂ may be incorporated into the synthesis of urea (15). Second, ¹³CO₂ may be incorporated into intermediate metabolites such as oxaloacetate or malate (15,24). Finally, ¹³CO₂ may participate in an exchange in the system of bone bicarbonate, contributing to increase its concentration in the osseous pool, with a consequent reduction in plasma flow. An experimental study on rats (25) demonstrated that, after 120 min of infusion, 7 to 10% of NaH¹⁴CO₂ is recovered in the skeleton. Considering the ¹³C incorporation into urea, we evaluated seric urea levels and detected no significant difference in ¹³CO₂ recovery fraction in expired air between groups with high and normal urea levels.

Since ¹³CO₂ is diluted in a pre-existing bicarbonate pool, we can rule out the possible interference of the basal serum bicarbonate level with the ¹³CO₂ recovery fraction in expired air. If the basal bicarbonate value is elevated, ¹³C will be diluted in a larger pool and therefore the chance of its elimination in expired air may be lower than in cases with lower bicarbonate levels. No significant difference was detected between patients with low and high bicarbonate levels.

Other factors may interfere with ¹³CO₂ retention in the organism, such as moderate and prolonged physical exercise, obesity (26), critical patient status (13), and fasting (8,16,17,20,21) or postprandial (18,22,23) condition.

CO₂ production should also be considered to be a factor that may interfere with the ¹³CO₂ recovery fraction in expired air. Since this variable may be affected by several factors, especially alveolar ventilation and oxidative stress, we evaluated selected patients in terms of their VCO₂ values. Assuming that ¹³CO₂ is eliminated by the lungs, the alveolar minute ventilation may interfere with its elimination and consequently affect the ¹³CO₂ recovery fraction in expired air. Alveolar minute ventilation probably did not interfere with VCO₂ values since the ventilatory pattern remained stable and physiological throughout the study protocol. On the other hand, the body weight factor, which determines the amount of bicarbonate infused and the individual metabolism, may alter the VCO₂ value. On this basis, a lower amount of ¹³CO₂ elimination would be expected by patients with higher VCO₂. However, 2 patients presented VCO₂ above normal values (>200 mL/min), while 7 patients presented VCO₂ below normal values and the ¹³CO₂ recovery fraction in expired air was similar for both groups.

In the present study, the ¹³CO₂ recovery fraction was 0.68 ± 0.06 in septic patients receiving nutritional therapy. There was no statistically significant difference between ¹³CO₂ recovery fraction in patients with sepsis of different severity, according to the APACHE II index and sepsis score. The same occurred when we evaluated different groups selected according to age and gender. The variation (0.68 ± 0.06%) among the patients was 8.8%, demonstrating that the result was homogeneous for the population studied. This result is important because the population of septic patients is intrinsically heterogeneous but, in this context, the heterogeneity did not interfere with the present measurements.

The factor that probably contributed most to the ¹³CO₂ recovery fraction in expired air was the metabolic rate. Wolfe (17) reported an increase in recovery fraction from 0.78 to 0.98 during intense and prolonged exercise. The same increase was detected by Hoerr et al. (20) in response to diet, with a positive correlation between VO₂, VCO₂, real energy expenditure and ¹³CO₂ recovery fraction in expired air in adults. However, Tissot et al. (12) detected a positive, although weak, correlation between the recovery fraction and VO₂ and real energy expenditure (r = 0.55 for both, with P < 0.05) and found no correlation between the recovery fraction and VCO₂. On this basis, care should be taken in interpreting these data because of the risk of mathematical coupling between parameters that share common variables (27,28). This risk is higher for the correlation between the recovery fraction (RF) and CO₂ production (VCO₂): [RF = (VCO₂ x ¹³CO₂ Enr)/(F x 0.99)]. On the other hand, minute volume is a variable used to calculate both recovery fraction and VO₂. In the present study, there was a positive, although weak, correlation between minute volume and ¹³CO₂ recovery fraction in expired air (r = 0.58) and a better correlation between real energy expenditure and ¹³CO₂ recovery fraction in expired air (r = 0.62) and between VO₂ and ¹³CO₂ recovery fraction in expired air (r = 0.75). These findings suggest that the correlation between these variables (VO₂ and ¹³CO₂ recovery fraction in expired air) was not the result of simple mathematical coupling since the variable shared by the two parameters (minute volume) did not correlate with ¹³CO₂ recovery fraction in expired air. Since VO₂ is a variable related to survival (29), the ¹³CO₂ recovery fraction in expired air might play a similar role, a fact that was not demonstrated in the present study, perhaps owing to the small number of patients evaluated. This line of reasoning is particularly relevant if we assume that a lower ¹³CO₂ recovery fraction in expired air may be related to greater severity, i.e., that a lower ¹³CO₂ recovery may mean that ¹³CO₂ is being shifted to alternative metabolic pathways that may be activated in order to resolve the injury in question.

However, the occurrence of incomplete ¹³CO₂ recovery in expired air has not been fully explained because there are no consistent data documenting in which organic compartment the isotope is lost. It is possible that a significant portion of the isotope is incorporated through alternative metabolic pathways that may function in different manners according to the pathological condition involved. Studies are needed to elucidate the values of the recovery fraction in different groups according to age, nutritional therapy and specific pathological conditions in order to better interpret the oxidation of substrates. On this basis, we can use an additional path, little explored until now, to alter the prognosis, to validate new treatment modalities and perhaps to improve the survival of patients with sepsis and septic shock.

12. Tissot S, Delafosse B, Normand S, Bouffard Y, Annat G, Viale JP, et al. Recovery of [¹³C]bicarbonate as respiratory ¹³CO₂ in mechanically ventilated patients. Am J Clin Nutr 1993; 57: 202-206.

Address for correspondence: M.A. Martins, Centro de Terapia Intensiva, HC, FMRP, USP, Av. Bandeirantes, 3900, 14049-900 Ribeirão Preto, SP, Brasil. Fax: [phone]. E-mail: [e-mail]

Research supported by the Pró-Reitoria de Pesquisa da Universidade de São Paulo, and Fundação de Amparo ao Ensino, Pesquisa e Assistência, Hospital das Clínicas, Faculdade de Medicina de Ribeirão Preto, USP, and FAPESP. Received October 25, 2007. Accepted May 30, 2008.

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Correspondence and Footnotes

Publication Dates

• Publication in this collection
  14 Aug 2008
• Date of issue
  July 2008

History

• Accepted
  30 May 2008
• Received
  25 Oct 2007