Postoperatively altered total clearance of ascorbic acid in surgical tumour patients

Background: Postoperatively lowered ascorbic acid (AA) plasma concentration is postulated to be due to an increased perioperative activity (radical scavenging activity, cofactor for enzymes, etc) and consumption in response to surgical trauma. Objective: To test this hypothesis by measuring total clearance (Clto t) of AA in plasma. Any alteration in AA consumption would be due to an altered Cltot of AA in plasma. Unaltered postoperative Clto t would exclude the postulated theory of an increased perioperative AA consumption. Design: The Clto t of AA in plasma was calculated for 17 tumour patients undergoing major maxillofacial surgery on the  rst and third postoperative days compared with the preoperative values after bolus injection of 4 mg kg 1 body weight AA was given intravenously. Blood samples were taken before and 15, 30, 45, 60 and 120 min after injection. AA in plasma was analysed by high-performance liquid chromatography. Results: The concentration of Clto t [median (25%:75% percentile)] was 10.2 (7.9:12.5) l h 1⁄4 1 preoperatively. On the  rst and third postoperative days [median: 17.6 (10.9:27.9) and 15.3 (12.1:30.5) l h 1⁄4 , respectively] the Clto t was signi cantly increased (pB0.05). Conclusions: Clto t was signi cantly increased in postoperative patients compared with preoperative values. These results are consistent with the postulated increase in perioperative AA consumption.


Introduction
A postoperatively lowered plasma concentration of ascorbic acid (AA) has been shown previously (1)(2)(3)(4)(5) and is interpreted as part of oxidative stress.Oxidative stress is de ned as an imbalance of increased pro-oxidants (radicals, etc.) and reduced antioxidants (vitamins, etc.) (6).It is postulated that the reduced antioxidants are the result of increased perioperative AA activity (radical scavenging activity, cofactor for enzymes, etc.).Increased perioperative AA activity would be accompanied by altered pharmacokinetics of AA.The term AA activity is used in this context to describe the clearance of AA from blood (total clearance¾ Cl t o t ).Cl to t is in uenced by the renal (Cl ren a l ) and metabolic (Cl m et a ) AA consumption.Therefore, AA activity is not identical to AA consumption.
At present, no data on AA clearance in postoperative patients are available.To investigate this, the preoperative and postoperative Cl t o t of AA were calculated.Values of preoperative and postoperative Cl to t that were not signi cantly different would exclude the postulated increased perioperative AA activity.

Patients
The investigation was carried out according to the recommendations for clinical trials in humans in the D eclaration of H elsinki and was approved by the Ethics Committee of the U niversity of M ainz.
Seventeen patients (62 9 13.3 years old, body mass index 22.8 9 3.2 kg m ¼ 2 ) were hospitalized for major maxillofacial tumour operations (12 for epithelial carcinoma, 3 for squamous cell carcinoma and 2 for basal cell carcinoma).Ten patients had preoperative radiotherapy and:or chemotherapy until some weeks before hospitaliza-tion, whereas seven patients had no neoadjuvant therapy.The average duration of operation was 525 9 138 min (mean 9 SD ).All patients were admitted to the intensive care unit (ICU ) postoperatively.N one of the patients showed any postoperative complications within the study period.Patients with at least one of the following factors were excluded from the study: pregnant patients, not fully mentally capable patients, and patients with a known history of allergic reactions to one of the substances in the AA ampoules, urethrolithiasis, ureterolithiasis, haemochromatosis, thalassaemia or any other tendency to haemolytic reactions.

Experimental pr ocedure
All patients were studied on the day before operation (P) and on the rst (D 1) and third (D 3) postoperative days.D uring each investigation blood samples were taken as baseline values of AA.Subsequently, a bolus of 4 mg kg ¼ 1 body weight AA was given intravenously.As Levine et al. (7) described in their depletion -repletion study, these amounts are sufcient to in uence pharmacokinetics without major adverse effects.Blood samples were taken 15, 30, 45, 60 and 120 min after bolus injection.A previously published method was used to analyse AA in plasma (8).In brief, all samples were collected in tubes with 1.6 g l ¼ 1 ethylenediaminetetra -acetic acid (ED TA) as anticoagulant.D uring the transport of the specimen to the laboratory they were stored on ice in darkness.Plasma was obtained by centrifugation, and cold 10% perchloric acid containing 1% metaphospho ric acid in brown microcentrifuge tubes was added for deproteinization.All samples were kept at 4°C for 60 min to complete the deproteinization process, followed by centrifugation.Subsequently, a mobile phase was added (1:1), and the samples were centrifuged, ltered and stored in liquid nitrogen until the analysis was carried out.All samples were analysed in duplicate by high-performance liquid chromatography with ultraviolet detection.
The Cl t o t of AA after bolus injection was then investigated.Cl t o t is de ned as dose:area under the curve (AU C).The AU C was obtained directly from the data (semi-log trapezoidal rule) above baseline and after extrapolation of the data to the moment of bolus injection.The residual AU C was calculated according to P6:k, where P6 is the last measured plasma concentration and k is the elimination rate constant.

Statistical analysis
Parameters were evaluated for normality using the Shakiro -Wilk test.Differences between postoperative parameters and preoperative baseline values were evaluated using the two-sided Student's t -test for parametric data and the Wilcoxon rank-sum test for skewed data.Sigma Stat ® was used during this statistical analysis.F or sample size determination the standard deviation of the AU C differences (D AU C of P and D 1) were calculated.The preoperative AUC was calculated at 28 mg l ¼ 1 per hour.To increase the probability that the relative difference was clinically relevant, the relative difference was set to 50% of preoperative AU C (14 mg l ¼ 1 per hour).The level of probability was set to 5% and b-error to 10%.U nder these conditions a minimum of 12 patients needed to be included in the study to achieve the necessary statistical power.

A rea under the curve and total clearance of ascorbic acid
The preoperative AU C (P) (29.6 9 15.3 mg l ¼ 1 per hour) and postoperative AU C (D 1) (14.2 9 14.7 mg l ¼ 1 per hour) was calculated for the determination of the sample size and Cl t o t .

Discussion
Postoperatively low concentrations ( B 30.2 mmol l ¼ 1 ) of AA in plasma have been described previously (9).Low AA concentrations are common in ICU patients (4,10) and the in uence of low AA concentrations on the onset of postoperative com- concentration is clinically relevant, the underlying mechanism is not totally clear.
Previously, reduced postoperative AA concentration in plasma was discussed as being a result of perioperative haemodilution.Several studies attempted to exclude haemodilution as a factor by the documentation of the haematocrit in plasma (13) or AA concentration in the buffy layer (1).The haematocrit or haemoglobin level is a useful marker to exclude haemodilution in patients without parenteral administration of blood products (e.g.myocardial infarction), but in perioperative situations, where patients may be given blood products, the haemoglobin is only a marker for an adequate substitution (corrected haemoglobin concentration) and not for haemodilution.
D espite the absence of any pharmacokinetic studies that de nitely reject the haemodilution theory and show a perioperative increase in the metabolic activity of AA, in the recent years the low postoperative AA plasma concentration has been postulated to be the result of increased consumption of AA due to non-nutritive effects (cofactor for enzymes, radical scavenging activity, etc.) in response to surgical trauma.To test this hypothesis the Cl t o t was calculated preoperatively and postoperatively.Any alteration in metabolic or renal AA consumption would lead to an alteration in Cl t o t .Preoperative and postoperative Cl t o t values that were not signi cantly different would exclude the postulated theory of increased perioperative AA consumption.plications in ICU patients has been discussed (11,12).Although it seems that the low AA plasma In this study a preoperative Cl t o t of 10.2 l h ¼ 1 was calculated.Similar clearance was reported by Levine et al. (7) in healthy volunteers.Postoperatively, a signi cant increase in Cl t o t was seen on the rst and third postoperative days.In addition, the postoperatively altered AA clearance seemed to be independent of preoperative AA plasma values.Even low preoperative AA plasma concentrations led to increased postoperative Cl to t in this study.
In general, Cl to t is in uenced by AA metabolism (Cl m eta ) and elimination (Cl ren a l ).In theory, the increased Cl to t seen here could be an effect of increased AA clearance by the kidney (Cl ren a l ).
The Cl ren a l is known to be dependent on the plasma concentration of AA.AA is actively excreted in urine until a threshold value is reached at the plasma concentration (14).In this study, preoperative and postoperative AA values in plasma were below the reference range and not signicantly different.Therefore, an unchanged renal clearance was postulated in these postoperatively uncomplicated patients on the rst and third postoperative days.
These data show an increased postoperative total clearance of AA in the blood compared with preoperative values.It could not be shown from the data whether the rise in Cl to t was an effect of increased renal function (Cl ren al ) or metabolic function (non-nutritive effects of AA) (Cl m et a ).F urther studies are necessary to differentiate between these functions.

Fig. 1 .
Fig. 1.Total clearance (Cl t ot ) of ascorbic acid in plasma preoperatively and on the rst and third postoperative days.Data are presented as medians with 25% and 75% percentiles.

Table 1 .
Area under the curve (AUC) and total clearance (Cl to t ) of ascorbic acid in plasma preoperatively and on the rst (D1) and third (D3) postoperative days