Are the enzymatic methods currently being used to measure bronchoalveolar lavage bile salt levels fit for purpose?

doi:10.1016/j.healun.2012.12.008

The Journal of Heart and Lung Transplantation

Volume 32, Issue 4, April 2013, Pages 418-423

https://doi.org/10.1016/j.healun.2012.12.008 Get rights and content

Background

Microaspiration after gastroesophageal reflux has been implicated in the chronic loss of allograft function in lung transplant patients. Bronchoalveolar lavage fluid (BALF) assessment for pepsin and bile salts is a common method to document reflux and aspiration. Clinically used methods for bile salt analysis include tandem mass spectrometry and diagnostic enzymatic kits designed to measure bile salts in serum. In clinical research, the enzymatic kits have been commonly used for BALF assays in lung transplant recipients, with reports of detection limits of 0.2 μmol/liter, and the levels used to inform clinical decisions. This study assessed the sensitivity of detection by 2 enzymatic assay kits compared with tandem mass spectrometry.

Methods

These 2 kits were used to measure (1) the absorbance changes for 0 to 50 μmol/liter bile salts, (2) levels in gastric juice (10–10,010 μmol/liter), and (3) bile salt levels of 40 BALF samples that were also measured using tandem mass spectrometry (0.01–1.19 μmol/liter). Measurements of pH/impedance were done in 14 of 15 patients.

Results

Neither kit had detection limits as low as claimed in previous BALF studies. The kits could be made more sensitive with a longer incubation time, (5 μmol/liter). All patients had detectable lavage bile acids using mass spectroscopy, 71% had pathologic distal gastroesophageal reflux, and 43% had pathologic proximal reflux.

Conclusions

The enzymatic kits are not sensitive enough for use in situations where bile salt levels are much below 5 μmol/liter, which is the case in BALF. In addition, reports in the literature of levels significantly below 5 μmol/liter need reassessing. Tandem mass spectrometry with a lower limit of detection of 0.01 μmol/liter should be the method of choice.

Section snippets

Materials and methods

Two spectrophotometric kits used to assess BALF and reported in the lung transplant literature were evaluated:

Kit 1: Bio-Quant Labs, San Diego, California, or Trinity Biotech PLC, Co Wicklow, Ireland, and
Kit 2: Alere San Diego Inc, San Diego, California, or Bio-Stat Diagnostics Systems, Cheshire, United Kingdom.

Both kits rely on the action of 3α-hydroxysteroid dehydrogenase (3α-HSD) releasing reducing equivalents from bile acids generating reduced NAD, which is measured directly

Kit 1

Kit 1 is as used by the groups reporting bile salt levels in lung lavages.5, 9 The formed coenzyme nicotinamide adenine dinucleotide hydrogen (NADH) further reacts with nitro tetrazolium blue in the presence of diaphorase enzyme to form a formazan dye, which absorbs at 540 nm. As seen in Figure 1A, 35 μmol/liter bile salts produces a change in absorbance of 0.046, diverted from linearity at the lower concentrations of 15 down to 2 μmol/liter, and concentrations of 6, 4, and 2 μmol/liter could

Discussion

BALF bile salt levels have been used to contribute to the clinical decision of whether lung transplant recipients receive fundoplication, and the above spectrophotometric methods are commonly used and reported for such assays. We found that BALF bile salts were detectable and quantifiable in lung transplant patients by tandem mass spectroscopy but not by the spectrophotometric assays, even when the assays were modified to improve sensitivity. Assessments on detection levels of the enzymatic

Disclosure statement

This study was funded by the Biotechnology and Biological Sciences Research Council, the European Society for Organ Transplantation, the British Lung Foundation, and the Medical Research Council.

None of the authors has a financial relationship with a commercial entity that has an interest in the subject of the presented manuscript or other conflicts of interest to disclose.

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Cited by (16)

Airway pepsinogen A4 identifies lung transplant recipients with microaspiration and predicts chronic lung allograft dysfunction
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Aspiration is a known risk factor for adverse outcomes post-lung transplantation. Airway bile acids are the gold-standard biomarker of aspiration; however, they are released into the duodenum and likely reflect concurrent gastrointestinal dysmotility. Previous studies investigating total airway pepsin have found conflicting results on its relationship with adverse outcomes post-lung transplantation. These studies measured total pepsin and pepsinogen in the airways. Certain pepsinogens are constitutively expressed in the lungs, while others, such as pepsinogen A4 (PGA4), are not. We sought to evaluate the utility of measuring airway PGA4 as a biomarker of aspiration and predictor of adverse outcomes in lung transplant recipients (LTRs) early post-transplant.
Expression of PGA4 was compared to other pepsinogens in lung tissue. Total pepsin and PGA4 were measured in large airway bronchial washings and compared to preexisting markers of aspiration. Two independent cohorts of LTRs were used to assess the relationship between airway PGA4 and chronic lung allograft dysfunction (CLAD). Changes to airway PGA4 after antireflux surgery were assessed in a third cohort of LTRs.
PGA4 was expressed in healthy human stomach but not lung. Airway PGA4, but not total pepsin, was associated with aspiration. Airway PGA4 was associated with an increased risk of CLAD in two independent cohorts of LTRs. Antireflux surgery was associated with reduced airway PGA4.
Airway PGA4 is a marker of aspiration that predicts CLAD in LTRs. Measuring PGA4 at surveillance bronchoscopies can help triage high-risk LTRs for anti-reflux surgery.
Commentary: Gastroesophageal reflux and lung allograft dysfunction: Need to improve detection and clinical reporting
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2020, Transplantation Proceedings
Citation Excerpt :
Clearly this determination is critical for generating the binary classification calculations reported in the Results section. Furthermore, citing differences in enzymatic techniques, some investigators have argued that the bile salt assay used in this study is insensitive [30]. However, more recently, other reports have emerged validating the same technology used here [12,31].
Gastroesophageal reflux (GER) in recipients of lung transplant (LTX) is associated with chronic allograft rejection, presumably via microaspiration that damages airway epithelium. Most LTX programs perform a single post-LTX esophageal study to evaluate for GER; the efficacy of this test is unclear.
Patients with 1 year of post-LTX follow-up, including routine bronchoscopies with bronchoalveolar lavage fluid (BALF) samples as well as high-resolution esophageal manometry and pH probe monitoring (HREMpH), were evaluated. BALF samples were analyzed with competitive enzyme-linked immunosorbent assay to detect bile salts, which are indicative of aspiration. These results were compared to results of HREMpH studies post LTX.
Ninety BALF samples were analyzed for bile salts and acted as disease positive for this evaluation. Of the 13 HREMpH cases, 8 were positive for GER, but only 3 were positive for bile salts via assay. Of the 5 HREMpH-negative cases, 2 experienced aspiration. A solitary HREMpH study had 60.0% sensitivity and 37.5% specificity with positive and negative likelihood ratios: 0.96 and 1.07, respectively.
Microaspiration appears to be an intermittent phenomenon, and HREMpH screening poorly correlates with BALF evidence of aspiration; which may not be adequate. As aspiration detection is crucial in this population, further analysis is warranted.
Determination of bronchoalveolar lavage bile acids by solid phase microextraction liquid chromatography-tandem mass spectrometry in combination with metabolite profiling: Comparison with enzymatic assay
2014, Journal of Chromatography A
Citation Excerpt :
This method is non-specific and may be subject to cross-reaction with other constituents that are chemically similar to BAs. As this enzymatic assay was originally designed for determination of BAs in serum samples, a recent study by Parikh et al. [6] evaluated the sensitivity of this method for measurements of BAs levels in BALF, which are significantly lower than those found in serum. Results have demonstrated that the enzymatic kits are not sensitive enough for determination of BAs levels in BALF samples.
A thin-film solid-phase microextraction (SPME) method coupled to liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis was developed for high-throughput determination of bronchoalveolar lavage bile acids. The proposed method was validated according to the bioanalytical method validation guidelines. LOQ and LLOQ were 0.007 and 0.02 μmol/L, respectively. The accuracy and the precision were <7 and <4%, respectively. The performance of the proposed method was also compared with an optimized enzymatic cycling assay. Results showed a weak correlation between total concentration of bile acid (BAs) obtained with enzymatic assay and cumulative concentration of specific BAs determined with SPME-LC-MS/MS. This discrepancy was probably due to the presence of other BAs in bronchoalveolar lavage fluid (BALF) samples. Metabolites profiling of BALF samples extracts using a high-resolution mass spectrometer (HRMS) revealed the presence of additional BAs, which were not included in the proposed method. After considering these additional BAs, a strong correlation was found between the LC-MS method and the enzymatic assay. Unsupervised statistical analysis conducted on HRMS data also showed clear separation within BALF samples, depending on the presence of BAs and other lipids. SPME-LC-MS-based metabolites profiling may provide additional information for diagnosis occurrence and severity of gastric reflux/aspiration in lung transplant patients.
BTS clinical statement on aspiration pneumonia
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Pepsin properties, structure, and its accurate measurement: a narrative review
2022, Annals of Esophagus

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Are the enzymatic methods currently being used to measure bronchoalveolar lavage bile salt levels fit for purpose?

Background

Methods

Results

Conclusions

Section snippets

Materials and methods

Kit 1

Discussion

Disclosure statement

Chest

Am J Transplant

J Thorac Cardiov Sur

Chest

Chest

Am J Transplant

Pepsin, a biomarker of gastric aspiration in lung allografts: a putative association with rejection

Am J Respir Crit Care Med

Pepsin like activity in bronchoalveolar lavage fluid is suggestive of gastric aspiration in lung allografts

Thorax

A call for standardization of antireflux surgery in the lung transplantation population

Transplantation

Screening of newborn infants for cholestatic hepatobiliary disease with tandem mass spectrometry

Brit Med J