Automated enzymatic assays for the determination of intestinal permeability probes in urine. 2. Mannitol

Clinica Chimica Acta, 183 (1989) 163-170

163

Elsevier

CCA 04504

Automated enzymatic assays for the determination of intestinal permeability probes in urine. 2. Mannitol Peter G. Lunn ‘, Christine

A. Northrop

’ and Andrew

J. Northrop

2

’ Dunn Nutritional Laboratory, Downham’s Lane, Cambridge and 2 AFRC Institute of Animal Physiology and Genetics, Babraham, Cambridge (UK) (Received 5 November 1989; revision received 3 April 1989; accepted 10 April 1989) Key words: Intestinal permeability; Mannitol; Automated enzyme assay

A need for a simple method for the determination of marmitol in urine has arisen because of the use of this monosaccharide in intestinal permeability tests. A rapid spectrophotometric assay for mannitol is presented based on the use of the bacterial enzyme mannitol dehydrogenase. The technique has been automated for use on the Cobas-Bio (Roche) centrifigal analyser. The assay has been shown to be highly specific for mannitol and was not affected by high concentrations of glucose, lactose or lactulose in samples. Within assay coefficient of variation was in the range 0.3 to 1.1% and 0.6 to 2.4% between assays. The technique represents a significant improvement in terms of time, simplicity and precision on existing methods.

Introduction

At least three different monosaccharides, xylose, rhamnose and mannitol have been used in the assessment of intestinal permeability. However, each probe molecule gives a different result both in terms of recovery over a 5-6 h collection and when compared to disaccharide excretion. Xylose values tend to be higher than the other two and this is now known to be because its uptake into the gut occurs least partially via a facilitated transport mechanism [l]. Rhamnose has been extensively used in permeability measurements but tends to give lower results than either xylose or mannitol. Although its absorption across the intestinal mucosa Correspondence to: P.G. Lunn, DUM Nutritional Laboratory, bridge CB4 IXJ, UK.

0009-8981/89/%03.50

Downham’s Lane, Milton Road, Cam-

0 1989 Elsevier Science Publishers B.V. (Biomedical Division)

164

seems entirely passive, recent studies have shown that its excretion in the urine is significantly delayed [2]. This result suggests that its distribution volume within the body may be greater than total extracellular water or that some reabsorption of the sugar occurs in the kidney. In either case the reduced excretion is clearly a complicating factor and makes its use as a permeability probe less desirable. Manmtol however does not suffer from these disadvantages and in an extensive study in man it was demonstrated that its uptake, internal distribution and excretion fit the criteria for use in passive permeability measurements [3]. Moreover, manmtol handling within the body was shown to be closely similar to that of lactulose, the disaccharide used most frequently in permeability tests. From this consideration it would seem that mannitol would be the monosaccharide probe of choice, however problems arise in its measurement. Currently available methods of assay are either highly non-specific [4] or involve chromatographic separation of urinary sugars, often after derivatisation, making accurate determination difficult and time consuming [5-71. Faced with a need to assay many hundreds of samples from permeability tests performed in epidemiological studies, we have developed an automated enzyme technique for mannitol which can be used on urine samples without prior preparation. The method makes use of a bacterial enzyme, mannitol dehydrogenase which catalyses the reaction: manmtol + NAD + fructose + NADH

Materials and methods

Mtitol dehydrogenase (EC 1.1.1.67) is not available extracted from a culture of the bacterium Leuconostoc (NCTC) 6992 equivalent to ATCC 9135 obtained from Station, Aberdeen Scotland). The growth of the bacterium partial purification of the enzyme were based on procedures [81.

commercially and was mesenteroides (NCIB NCIB, Torry Research and the extraction and described by Yamanaka

Bacterial culture The growth medium contained peptone, 10 g/l; yeast extract, 2 g/l; D-glucose, 0.056 mol/l; sodium acetate, 0.12 mol/l; magnesium sulphate, 0.9 mmol/l; sodium chloride, 0.017 mmol/l and manganese sulphate, 0.009 mmol/l. 8 ml of this medium was inoculated with the bacterium and incubated overnight at 30 o C. This culture was transferred to 400 ml of the same medium which was incubated for a further 24 h at 30 o C. The entire volume was then used to inoculate 20 1 of medium which was incubated with aeration for 18 h at 30” C. Cells were harvested by centrifugation at room temperature using a zonal rotor at 10000 X g with a flow rate of about 10 l/h. After two washings in 10 vol of phosphate-mercapthoethanol buffer (50 mmol/l) potassium phosphate containing 1 mmol/l mercaptoethanol, pH 7.0), cells were resuspended in 5 vol of this buffer, cooled to 4O C in an ice bath and disrupted by sonication (3 x 30 s treatment). On some occasions the bacteria

165

were resistant to this treatment and at such times a Dyno Switzerland) was used to achieve cell breakage.

Mill (W. Bachofen,

Basle,

Enzyme extraction Unless otherwise stated, the extract was maintained at 0-4°C throughout the purification procedure. 100 ml of the phosphate-mercaptoethanol buffer was added to the disrupted cells and the extract centrifuged at 10000 X g for 20 min. The precipitate was discarded. Protamine sulphate solution (20 g/l, added at 4.6 ml of solution per 100 ml of extract), was added dropwise and with slow stirring to the supernatant. After standing for 1 h, the extract was centrifuged (10 000 x g, 20 min), and the precipitate discarded. Ammonium sulphate, (with reduced heavy metal content, Sigma) 71 g/100 ml was added to the protamine sulphate supematant to precipitate all the protein in the extract. The mixture was stirred to dissolve the ammonium sulphate and then allowed to stand for 20 min before centrifugation, (10000 X g, 20 min). The supematant was discarded, the precipitate dissolved in 20 ml of phosphate-mercaptoethanol buffer and dialysed overnight against 2 1 of the same buffer. Ammonium sulphate fractionation Following dialysis, ammonium sulphate, (2.87 g/10 ml dialysate) was added and after standing for 20 min the preparation was centrifuged (10000 X g/20 min) and the precipitate discarded. More ammonium sulphate was then added (1.04 g/10 ml of dialysate volume) to the supernatant which after standing for 20 min was centrifuged (10000 X g, 20 min). The precipitate, a bright yellow colour, was retained, dissolved in 10 ml of phosphate-mercaptoethanol buffer and dialysed overnight against 1 litre of the same buffer. Acetone fractionation The dialysed ammonium sulphate fraction was adjusted to pH 6.0 with 0.02 mol/l acetic acid. Acetone, (4.6 ml/10 ml of extract) previously chilled to - 20 ’ was added dropwise with gentle stirring and after standing for 5 mm the precipitate was removed by centrifugation (10000 X g, 10 min at - lO”C), and discarded. More acetone, (4.4 ml/10 ml supernatant) was added as before and after 10 min standing, the precipitate was collected by centrifugation, (10000 x g, 10 min at - 10°C) and dissolved in 10 ml phosphate-mercaptoethanol buffer. Ammonium sulphate, (5.15 g/10 ml) was then added to precipitate the enzyme and after standing for 20 min the solution was centrifuged, (10000 x g, 20 mm). The precipitate was dissolved in 10 ml of phosphate-mercaptoethanol buffer and dialysed overnight against 1 litre of buffer. After dialysis, l-ml portions of the enzyme preparations were frozen at - 20 o C until use. No apparent deterioration in activity occurred during storage for at least 9 mth at this temperature. Standardisation of enzyme The activity of the purified enzyme was assessed using the standard assay conditions described below. 20 ~1 of 0.75 mol/l mannitol was used (as sample) and

166

the enzyme preparation diluted (usually by about 1: 1000) to give an absorbance change of < O.l/min. One unit of enzyme is defined as the amount required to generate 1 pmol of NADH/min under standard assay conditions. The enzyme was diluted in phosphate-mercaptoethanol buffer to about 50 U/ml. In practice 50 g of bacteria yield about 3 000 U of enzyme, sufficient for > 3 000 assays. The specific activity of the enzyme was about 16 U/mg. Analysis

Glycine buffer (0.4 mol/l) containing 0.5 mol/l hydrazine was prepared by dissolving 3 g of glycine (Sigma) in 50 ml of deionised water and adding 2.5 ml of an 85% aqueous solution of hydrazine hydrate (Sigma). The buffer was adjusted to pH 8.6 with dilute hydrochloric acid, made up to 100 ml and stored in a dark bottle. Immediately before use NAD (Boehringer) was added to the buffer solution at 75 mg/lO ml. As only 40 ~1 of this reagent is used for each determination on the centrifugal analyser, 10 ml of solution is sufficient for over 200 analyses. Approximately 100 ~1 of urine was placed in a Cobas-Bio (Roche) sample cup and loaded on to the analyser. The assay was calibrated using three mannitol standards, normally 250, 500 and 1000 mg/l (1.37, 2.74 and 5.49 mmol/l). The analyser was programmed as shown in Table I. During operation, 20 ~1 of sample (or standard) plus 10 ~1 of water, (as washout) was added to 40 ~1 of buffer-NAD reagent. After mixing, the absorbance at 340 nm was recorded and 15 yl of mannitol dehydrogenase added. The rise in absorbance at 340 nm was followed for 5 min by

TABLE I Programme parameters for mannitol assay on the Cobas-Bio (alpha) + Mamritol 1 2 3 4 5 6 I 8 9 10 11 12 13 14 15 16 17 18 19

units Calculation factor Standard 1 Standard 2 Standard 3 Limit Temperature ( a C) Type of analysis Wavelength (nm) Sample volume ( p 1) Diluent volume (~1) Reagent volume (~1) Incubation time (xc) Start reagent volume (pl) Time of first reading (xc) Tune interval (set) Number of readings Blanking mode Printout mode

mg/f 0 250 500 1000 1000 40 6 340 20 10 40 10 15 0.5 30 10 1 1

which time a plateau value was reached. The change in optical density was a measure of the amount of NAD converted to NADH and thus directly proportional to the mannitol content of the sample. The reaction was carried out at 40” C. Results

The relationship between mannitol concentration and the rise in optical density at 340 nm is shown in Fig. 1. Between mannitol concentrations of 62.5 mg/l (0.34 mmol/l) and 1000 mg/l (5.49 mmol/l), the response is almost linear but curves slightly at both low and high values, the latter effect being probably due to product inhibition of the enzyme reaction. Nevertheless, assay precision is extremely good (Table II) with a mean interassay coefficient of variation below 1%. Between assay variability, though slightly larger is clearly well within acceptable limits. Table III demonstrates the ability of the assay to accurately measure mannitol following addition of the sugar to normal urines to give concentrations over the

2.0

/

1.6

0.4

I

250

Mannitol Fig. 1. Relationship

between

mannitol

I

500

concentration concentration

I

750

I

1000

(mg/l) and the change

in absorbance

at 340 nm.

168 TABLE

II

Inter- and intra-assay

precision

at different

concentrations

of manmtol

expressed

as CV

cv

Cone mmol/l

mg/m

n

Inter-assay

n

Intra-assay

1.37 2.74 5.49

250 500 1000

15 15 15

0.67 0.30 1.09

10 10 10

2.42 0.58 1.30

TABLE

III

Mean recoveries

of different

concentrations

of mannitol

following

addition

to five normal

Cone

Recovery

mmol/I

mg/I

Mean

SE

1.37 2.74 5.49

250 500 1000

103.1 99.8 99.6

0.7 0.6 0.4

TABLE

urines

(W)

IV

Determination

of mannitol

in the presence Observed:

Mann&o1 cone

of 10 g/l of other urinary

sugars

actual cone (W)

mmoI/I

mg/I

Fructose

Glucose

Lactulose

Lactose

1.37 2.74 5.49

250 500 1000

47.3 48.9 51.5

103.9 102.2 98.4

100.2 102.1 99.2

103.9 101.0 98.0

Mean

49.2

101.5

100.5

101 .o

TABLE

V

Specificity of the mannitol dehydrogenase of < 0.01% of the mannitol response Arabinose Fructose GaIactose Glucose Lactose Lactulose Maltose Raffinose Rbamnose Sorbose Sucrose

assay for mamritol.

Trehalose Turanose Xylose Adonitol Arabitol Dulcitol Erythritol Glycerol Inositol Sorbitol XyIitol

The compounds

listed showed

a reaction

169

range most frequently encountered during permeability assessments. Certainly in normal urines there was no evidence of any inhibitor mechanisms. In abnormal urine from patients however, significant amounts of other sugars in the urine could potentially interfere with the assay so this possibility has been investigated. Table IV shows the percentage of observed: actual concentrations of manmtol when assayed in very high concentrations, i.e. 10 g/l of four such sugars, fructose, glucose, lactulose and lactose. Of these, only fructose showed any sign of interference with the marmitol determination. The 50% reduction caused by fructose was expected as this sugar is the end product of the enzyme assay and will thus lower the result by a product inhibition mechanism. The specificity of the assay for manmtol has also been examined using a range of sugars at a 10 g/l concentration, Table V. None of the compounds tested gave a reaction > 0.01% of the mannitol response. The mannitol dehydrogenase enzyme appears to be very highly specific for its substrate. Discussion The enzymatic assay of mannitol has many advantages over previous measurement techniques, but the ease and speed of the determination is probably its most significant feature. Using a centrifugal analyser, > 100 estimations can easily be performed per hour and no prior extraction or treatment of the urine samples is required. This is clearly in marked contrast to the lengthy and complicated extraction, derivatisation and chromatographic procedures available at present. In addition, the increased speed of assay is associated with a very significant improvement in precision, the CV is very much lower than figures obtained with chromatographic techniques. Moreover, because the procedure is so straightforward there is little chance of errors being made. The improved speed and precision do not result in any loss of specificity because the partially purified mannitol dehydrogenase is so highly specific for its substrate. Very high concentrations of glucose, lactulose and lactose were entirely without effect on the assay, and with the exception of fructose there was no indication that other normal constituents of urine exerted any interference. Fructose, however, did lower the value obtained for mannitol in the assay but this must be expected as this is the product of the enzyme reaction and the effect is clearly one of product inhibition. Nevertheless, inhibition of the reaction was constant over the full range of mannitol concentrations, i.e. at a fructose concentration of 10 mg/ml all mannitol values obtained were about 50% of the actual figure. It is therefore possible to correct the mannitol reading according to the fructose concentration, (measured as described in the preceding paper). None of the other sugars tested showed any significant reaction in the system. Above a concentration of 1.0 mg/ml (5.49 mmol/l), the relationship between mannitol and absorbance at 340 nm becomes more markedly curved and samples giving values beyond this point should be re-assayed after reducing the sample volume to 5 ~1. This is a simple procedure on the Cobas-Bio and extends the useful range up to 4.0 g/l (22.0 mmol/l). Samples containing < 62.5 g/l (0.35 mmol,/l)

170

are however less easily dealt with as the assay does not work well at such very low concentrations. The probable explanation for this is that the enzyme reaction is reversible and although the pH optima are quite different, i.e. 8.6 for the forward reaction and 5.3 for the reverse, a significant amount of the latter reaction seems to occur at low substrate concentrations. Thus samples containing < 62.5 mg/l (0.35 mmol/l) mannitol are best concentrated by freeze-drying before assay. In practice however, samples with such low mannitol content are rarely encountered following permeability tests. A very close correlation between mannitol concentrations assessed by the enzymic assay and by “C-counting has been demonstrated in a series of permeability studies performed in normal, obese and fasting subjects, a study with involved the estimation of marmitol in plasma as well as urine [3,9]. Other investigations in which the assay has been used are reported in the preceding paper [lo]. At present, mannitol dehydrogenase is not available commercially, but both the bacterial culture and the extraction procedure are quite straightforward and have invariably proved successful if the protocol is carefully followed. A 50-l growth of bacteria will yield sufficient enzyme for several thousand assays using the Roche Cobas-Bio analyser. The ability to determine urinary levels of lactose, lactulose and mannitol by fast, accurate, automated enzymatic techniques has allowed us to perform very large numbers of permeability estimations with the minimum of time and expense. Their adoption will clearly allow this useful test of small bowel integrity and function to become a routine clinical tool. References 1 Menzies IS. Transmucosal passage of inert molecules in health and disease. In: Intestinal absorption and secretion. Falk Symposium 36. London: MTP Press, 1983;527-543. 2 Maxton DB, Bjamason I, Reynolds AP, Catt SD, Peters TJ, Menzies IS. Lactulose, *‘Cr-labelled EDTA, t-rhamnose and polyethyleneglycol 500 as probe markers for assessment in vivo of human intestinal permeability. Clin Sci 1986;71:71-80. 3 Elia M, Behrens R, Northrop C, Wraight P, Neale G. Evaluation of mamritol, lactulose, “0-1abelled EDTA as markers of intestinal permeability in man. Clin Sci 1987;73:197-204. 4 Corcoran AC, Page IH. A method for the determination of manmtol in plasma and urine. J Biol Chem 1943;170:165-171. 5 Menzies IS, Mount JN, Wheeler MJ. Quantitative estimation of clinically important monosaccharides in plasma by rapid thin layer chromatography. Ann Clin Biochem 1978;15:65-76. 6 Laker MF, Mount JN. Manmtol estimation in biological fluids by gas-liquid chromatography of trimethylsilyl derivatives. Clin Chem 1980;26:44-443. 7 Hamilton I, Hill A, Bose B, Bouchier IAD, Forsyth JS. Small intestinal permeability in pediatric clinical practice. J Pediatr Gastroenterol Nutr 1987;6:697-701. 8 Yamanaka K. D-Manmtol dehydrogenase from L.euconoctoc mesenteroides. In: Wood W, ed. Methods in enzymology, Vol. 41 (Part B). New York: Academic Press, 1975;138-142. 9 Elia M, Goren A, Behrens R, Barker RW, Neale G. Effect of total starvation and very low caloric diets on intestinal permeability in man. Clin Sci 1987;73:205-210. 10 Northrop CA, Lunn PG, Behrens RI-I. Automated enzymic assays for the determination of intestinal permeability probes in urine. 1: Lactulose. Clin Chim Acta 1989;in press.