Higher Levels Of Hair Ethyl Glucuronide in Patients With Decreased Kidney Function

ali.ma@wecistanche.com

Gudrun Høiseth, et al

Background:

Hair levels of ethyl glucuronide (EtG) are often used to differentiate social drinking from heavy drinking. Patients with decreased kidney function have delayed excretion of EtG, and increased incorporation into hair could be suspected. The aim of this study was to compare hair EtG levels in patients with decreased kidney function to those seen in healthy volunteers.

Methods: Twelve patients with renal disease were included. The levels of EtG in hair were adjusted to an estimated daily intake of ethanol (EDI) and compared to 21 previously published healthy individuals.

Results: The levels of hair EtG in the 12 patients ranged between < limit of detection and 134 pg/mg, and the EDI ranged between 0.1 and 12 g. The levels of EtG in hair were significantly higher in the patients compared to healthy volunteers (p = 0.009).

Conclusions: These preliminary results indicate that hair levels of EtG in a population of patients with decreased kidney function should be interpreted with caution.

Key Words: Alcohol, Biomarker, Ethyl Glucuronide.

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THE LEVELS OF ethyl glucuronide (EtG) in the hair are used as a marker of long-term alcohol use, and a cut-off value of 30 pg/mg in the proximal 3-cm hair segment has been suggested to detect a high daily intake of alcohol (above 60 g/d; Potgieter, 2000) during the last 3 months (Alt et al., 2000; Morini et al., 2009). So far, all published articles indicate that EtG in hair shows the best diagnostic sensitivity and specificity for the detection of chronic heavy alcohol consumption compared to other traditional ethanol (EtOH) biomarkers. Only hair treatment, such as dying or bleaching, could negatively interfere with EtG determination, while all the other parameters evaluated (age, gender, hair color, etc.) seem not to affect EtG reliability (Morini et al., 2010).

We have recently shown prolonged urinary detection times for EtG and EtS in patients with decreased kidney function(Hoiseth et al., 2012). As renal excretion is the major elimination route for EtG and ethyl sulphate (EtS), this implies that higher blood concentrations of EtG and EtS could be seen in this patient population compared to healthy volunteers, after an identical intake of alcohol. This situation was also observed in 1 single subject with kidney disease in a previous study (Hoiseth et al., 2009). Higher blood levels of EtG could result in more incorporation into hair and subsequently higher levels of EtG in this matrix. The aim of this study was therefore to investigate whether hair levels of EtG in patients with decreased kidney function differed from those seen in healthy volunteers.

MATERIALS AND METHODS

Study Protocol

Nondialyzed renal disease patients were recruited from the renal failure clinic at Akershus University Hospital, Lørenskog, Norway. Inclusion criteria were serum creatinine value >90 lmol/L in women and >100 lmol/L in men (Rustad et al., 2004), estimated glomerulus filtration rate (GFR) <60 mL/min/1.73 m2 (automatically calculated from creatinine using the modification of diet in renal disease formula including age and sex; Rule, 2007), and a diagnosis of decreased kidney function in the patient’s journal. Only patients having a moderate use of alcohol were included in our study that was approved by the National Committee for Research Ethics in Norway, the Norwegian directorate of health, and the local ethics committee at Akershus University Hospital. All patients signed informed consent before attending the study.

An estimated daily intake of EtOH (EDI; calculated on the basis of the timeline follow-back test) for the last 3 months was obtained by interviewing the patient's care about the type of beverage and amount consumed on a typical day. Information was also received from the patients’ medical record. This included diagnoses, serum creatinine value, and the estimated GFR, as objective measurements of the individual renal function. Values of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyl transferase (GGT) were also collected, as objective measures of possible alcohol overconsumption. Hair was collected from each patient by cutting a hair specimen as close as possible to the skin. The 3-cm proximal hair segment, roughly representing the last 3 months, was submitted for EtG analysis. The results from these patients were compared to 21 previously published healthy volunteers (Politi et al., 2006).

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Analytical Methods

EtG in hair was determined using a previously published method (Morini et al., 2006). Briefly, about 100 mg of hair was washed with 2 different organic solvents (dichloromethane and methanol), dried under nitrogen stream, and cut into small pieces (1 to 2 mm length segments). Samples were then soaked into 700 lL of deionized water with penta-deuterated ethyl glucuronide (D5-EtG) as internal standard and incubated overnight at room temperature. On the following day, they were ultrasonicated for 2 hours and centrifuged at 10,8009g. Finally, 8 lL were injected into the liquid chromatography-tandem mass spectrometry system. The chromatographic separation was carried out in reversed-phase, isocratic mode, using a mobile phase containing a very high water percentage (0.1% formic acid + 1% acetonitrile). The mass spectrometer was used in multiple reaction monitoring modes, and negative polarization and 2 different transitions were used for the identification of EtG and D5-EtG. A calibration curve ranging from 3 to 200 pg/mg was used for quantification purposes. The limit of detection (LOD) was 2 pg/mg and, the lower limit of quantification was 3 pg/mg.

Statistics

Differences between hair levels of EtG in patients and healthy volunteers were studied using a multiple linear regression analysis with the EDI as a covariate. The p-values for a difference between groups (adjusted for doses) are given. A p-value below 0.05 was considered significant. Correlation between hair levels of EtG and GFR or serum creatinine was assessed using Spearman’s nonparametric rank correlation test.

RESULTS

Twelve patients were included (8 men and 4 women). The median age was 64 years (range, 42 to 84), and the median body mass index was 26.2 kg/m2 (range, 18.9 to 29.4). All 3 criteria for decreased kidney function were present in all patients. The median serum creatinine value was 189 lmol/L (range, 130 to 670). The median estimated GFR was 29.2 mL/min/ 1.73 m2 (range, 8.0 to 54.0), and all subjects had a diagnosis of decreased renal function in their medical record. All subjects had ALT, AST, and GGT within the normal range ( <=50 U/L for ALT and AST, <= 60 U/L for GGT; Klauke et al., 1993; Rustad et al., 2004; Szasz, 1974).

The levels of hair EtG in the 12 patients ranged between <LOD and 134 pg/mg, and EDI ranged between 0.1 and 12 g. The 2 highest values of hair EtG were seen in the patients with the most reduced kidney function. This is shown in Table 1. In the previously published 21 healthy volunteers used for comparison, the hair EtG ranged between <LOD and 35.4 pg/mg and EDI ranged between 2 and 60 g (Politi et al., 2006). The dose-adjusted levels of EtG in hair for the patients in the present study (n = 12) were significantly higher than for previously published healthy volunteers (n = 21; p = 0.009). This is shown in Fig. 1.

In the patients with decreased kidney function (n = 12), there was a significant correlation between the level of EtG in hair (corrected for EDI) and the GFR (Spearman’s ρ = 0.61, p = 0.036). The correlation between hair EtG (corrected for EDI) and serum creatinine was somewhat weaker and not statistically significant (Spearman’s ρ = 0.53, p = 0.076).

DISCUSSION

This study suggested that among social drinkers, levels of EtG in the hair are higher in at least some patients with decreased kidney function compared to healthy volunteers. The relation between hair EtG level and GFR also strengthened this assumption.

The finding of higher levels of EtG in hair in this patient group was expected from theoretical considerations, but to the authors' knowledge, has not been addressed earlier. In a healthy population, previous studies indicate that very few social drinkers have hair EtG values above the suggested cut off for heavy alcohol use (30 pg/mg; Alt et al., 2000; Appenzeller et al., 2007a; Politi et al., 2006; Pragst and Yegles, 2008; Yegles et al., 2004). The present population had EDI values way below the threshold for heavy drinking, but 3 patients still had EtG hair levels suggesting heavy alcohol consumption. One could further imagine that renal disease patients with higher intakes, but still within the definition of social drinking, more frequently could exceed the level of 30 pg/mg and incorrectly be classified as heavy drinkers. Also, in patients where a very low intake of alcohol is a requirement (e.g., in the case of kidney transplantation), the measurement of EtG levels in hair could lead to false conclusions of higher intakes.

The first weakness of this study is the small number of patients. This was a consequence of the size of the patient group with the actual clinical condition, and the general advice for this group to refrain from alcohol consumption. The present work must be considered as a preliminary report and larger studies are needed to confirm the results. On the other hand, the fact that the levels of hair EtG in such a limited number of patients were significantly higher compared to healthy volunteers could indicate that the difference is substantial. A second weakness of the present study is that the patients were consumers of small daily amounts of alcohol, resulting in hair EtG values below LOD for 7 of 12 participants. This could have been a consequence of the patients following clinical advice of reducing alcohol consumption because of the kidney disease. Underreporting of EDI is also a possibility. On the other hand, a validated questionnaire was used to minimize this source of error, and the same questionnaire was used in the previous study used for comparison. Also, the values of AST, ALT, and GGT within reference values at least indicated that there were no heavy drinkers among the patients. Further, we compared the results from the Norwegian patients in the present study to Italian healthy volunteers. This could represent an uncertainty with respect to interpretation, but previous reports indicate that different pigmentation does not affect the results of EtG in hair (Appenzeller et al., 2007b). Also, the same analytical method was used.

Considering the possible consequences of a false positive diagnosis of heavy drinking based on hair EtG in patients with kidney disease, further studies should be undertaken to gain more insight into the relationship between alcohol consumption, the degree of kidney failure, and hair EtG levels.

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ACKNOWLEDGMENT

The authors are grateful to Dr. Eirik Pettersen at Akershus University Hospital for assistance in recruiting the patients.

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