Determination of lactose malabsorption by breath analysis with gas

Biochemical Tests in Diseases of the Intestinal Tract: Their Contributions to Diagnosis, Management, and Understanding the Pathophysiology of Specific...
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mate reaction. Preliminary experiments show that this can be done by acylation.

In the analysis of serum samples, the heptane extraction was followed by a re-extraction to get rid of some disturbances from serum. Figure 2B shows a chromatogram from a serum sample with 20 ng of trimipramine per ml. Rectilinear standard curves were obtained in the range of 0-100 ng of trimipramine. T h e lowest amount t h a t could be determined with the present method was 5 ng in l ml of serum. T h e relative standard deviation a t the 80- and 20-ng level was 6 and 7% respectively. One important metabolite from the tertiary amines in the imipramine series is the N-demethylated one. Secondary amines form together with chloroformates the same carbamates as tertiary amines do. Therefore, it is essential t h a t the secondary amine be excluded before the chlorofor-

LITERATURE CITED (1) (2) (3) (4) (5)

(6) (7) (8) (9) (10)

P. Hartvig and J. Vessman, Anal. Lett., 7, 223 (1974). P. Hartvig and J. Vessman, J. Chromatog. Sci., 12, 722 (1974). I. C. Raiford and G. 0. Inrnan, J. Am. Chem. SOC.,56, 1586 (1934). A. C. Moffat and E. C. Horning, Anal. Lett, 3, 205 (1970). H. Brotell. H. Ehrsson. and 0. Gyllenhaal, J. Chromatogr., 78, 293 (1973). F. K. Kawahara, Anal. Chem., 40, 1009 (1968). A. Zlatkis and 6. C. Pettitt, Chromatographia,2, 484 (1969). S. B. Matin and M. Rowland, J. Pharm. Sci., 61, 1235 (1973). W. E. Wentworth and E. Chen, J. Gas Chromatogr., 5, 170 (1967). P. Hartvig and L. Maukonen, Farm. Aikak., 83, 141 (1974).

RECEIVED for review August 8, 1975. Accepted October 22, 1975.

Determination of Lactose Malabsorption by Breath Analysis with Gas Chromatography H. L. Gearhart,'

D. P. Base,* C. A.Smith,*

and

R. D. Morrison3

Oklahoma State University, Stillwater, Okla. 74074

J. D. Welsh and T. K. Smalley Department of Gastroenterology,University of Oklahoma, Health Science Center, Oklahoma City, Okla. 73 790

A method is presented for sampling and analyzing excreted HZ (

c m

I--

60

0

100

120

240

I

0

e 80

TIME (MIHI

-

Breath H2 concentration of Subjects 6, 7, and 8 vs. time after consumption of 5 ml reconstituted dry skim milk/kg body wt Figure 4.

40

0

io

20

30

40

50

60

70

90

80

100

CONCENTRATION O F H2 IPPMl

Figure 2. Helium ionization detector response vs. H2 concentration. Linear and quadratic least squares fits shown using standard mixtures

7a n9a 74 l y z e d ? 10 74 7 1 1 74 1 1 3 74

7 15 74 7 17 74

I-

n

40

80

120

‘60 TlhlE

200

240

280

DC

MIU

Figure 3. Breath H2 concentration for Subject 1 vs. time after consumption of 5 ml reconstituted dry skim milk/kg body wt on 5 alternate days

sponse to constant sample size vs. time was performed. I t was desired to evaluate the instrument and technique with respect to sources of variance including instrument drift, dilution effects in filling bags, and technician error in sampling and area triangulation calculations. Comparison of mean square values calculated from data given in Table I1 indicates that magnitude of variance between duplicates is on the same order as overall error. The four standard curves are essentially coincident ( F = 0.386), implying negligible drift. The coefficient of variation is relatively small (-1.5%). A least squares fit was used to determine coefficients with standard samples. Above 10 ppm, He detector response was seen to have significant quadratic curvature (see Figure 2) which is in agreement with previous work with the HID ( 1 7 ) .Subsequent samples with unknown concentrations of breath-Hs were analyzed, peak area triangulated, and ppm Hz calculated from predetermined coefficients. Diffusion loss rate studies were also done. Three randomly selected sample bags were filled with a standard mixture (50 ppm Hp/He) and analyzed a t intervals over a 24-hr period. Results are summarized in Table 111. Analysis of variance was done for H2 diffusion loss from sample bags vs. time. I t was apparent that sources of variance due t o diffusion loss as well as to differences between the three samples are insignificant for this time interval. Again, mag-

nitude of error between duplicates was minimal. As shown in Table IV, Subjects 1 through 5 all invariably had a maximum average breath Hp concentration of a t least 30 ppm within the test time interval. Results of the B H T for Subject 1 are shown in Figure 3. This represents typical behavior for B H T curves done on malabsorbers. Figure 4 is representative of B H T results obtained on absorbers where data are shown on three subjects (6 through 8), known to be lactose absorbers by L T T data (Table IV). They were tested using the same procedure as outlined for malabsorbers, but only for one day or two alternate days each. Data are also shown in Figure 5 for B H T results of a lactose malabsorber (Subject 1) in a time interval following ingestion of 5 ml/kg body wt reconstituted dry nonfat milk (lactose hydrolyzed). This test diet was analyzed by GC, and it was found to contain 8% unhydrolyzed lactose relative to the reconstituted skim milk used in previous tests. Analysis of variance (AOV), using a randomized block design, was performed to determine the magnitude and significance of individual response differences as well as day to day changes. I t was also desired to determine a reliable means of measuring the response in terms of excreted H2. Mean squares for all individuals on given test days and for the 5 test days computed for each individual (Table V) are shown in Table VI for each of the nine selected response

Table 11. Mean Areas (arbitrary units) for Duplicate Analysis of Three Standard Samples of Various Concentrations at Intervals over a 24-hr Period H , concentration, p p m Trial time. hr

9

50

97

Average0

0:oo

15.5 15.5 14.5 15.0 15.1

131.0 129.0 128.5 128.5 129.3

294.0 298.8 296.3 297.5 296.7

146.8 147.8 146.4 147.0

4:OO 8:OO 24: 00 Average a C.V. = 1.5%.

____

Table 111. Mean Areas (arbitrary units) for Duplicate Analysis of Contents from Three Sample Bags, Each Containing 50 ppm H, at Intervals over a 24-hr Period Sample Time, h r

1

2

3

Averagea

0:oo

131.0 128.5 126.0 127.5 128.5

127.5 128.5 125.0 129.0 127.5

130.0 130.0 129.0 130.0 129.8

129.2 129.0 126.7 128.8

4:OO 8: 00 24:OO Average a C.V.

=

1.6%.

ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976

395

"r *DAY#*( -DAY#2 )-----e

70

0

-,0

'

120

DAY X 3

- - - *DAY*4

I 180

I 240

I 300

TIME ( M I N I

Flgure 5. Breath H2 concentration for Subject 1 vs. time after consumption of 5 ml reconstituted 92% lactose-hydrolyzed, nonfat milk/ kg body wt on 4 alternate days

factors. As is shown in Table V, the initial (zero min) concentration of Hz was subtracted from all subsequent readings on a given test day in certain cases. Other response factors were calculated from all readings. In three other cases, no subtraction was made and absolute values were used instead. From comparison of calculated CV's (coefficient of variation), it is seen (Table VI) that more precision (minimum CV) is obtained if absolute values are measured, regardless of whether one picks the maximum H2 concentration, the average of 3 highest values, or the area under the concentration vs. time curve as a response factor. Since area calculations are relatively cumbersome for routine technician use, it is recommended that the 3 highest consecutive H2 concentration values taken between 60 and 180 min be averaged and the result used for a measure of individual response. Sampling a t 15-min intervals between 60 and 180 min following test diet consumption results in 9 samples collected, and requires 3 hr of the subject's time. Samples taken prior to or immediately following diet consumption may be discarded, but serve to familiarize the subject with the sampling technique. Examination of Tables V and VI indicates a significant difference among individuals' response. Table V shows Subject 1 averaged 76.8 ppm peak H2 values between 60 and 180 min following consumption of the test diet while Subjects 3 and 5 averaged near 45-50 ppm. T h e remaining two subjects gave intermediate values. Day effects are also evident but not as pronounced as individual effects. I t is interesting to note (Table V) that the mean breath H2 response increased gradually from Tuesday through Saturday and then decreased as the last test day was approached in all subjects considered. This observation could be due to various physiological and/or psychological effects, but a definite explanation cannot be given a t this time. Even though a day effect was evident, it did not interfere with conclusions drawn as t o whether or not subjects were determined t o be malabsorbers using the method outlined, Le., all had a rise of at least 30 ppm.

DISCUSSION Consideration of the results on the AOV study of detector response on several samples made u p and analyzed in an identical manner indicates clearly t h a t reproducible results may be obtained from the system described. Dilution effects introduced in filling the sample bags introduce negligible error. Since the detector was observed to give nonlinear response (peak area vs. HZ concentration), a nonlin396

ear least squares fit is recommended for the quantitative calibration curve technique. As a result of the diffusion loss rate studies on bags, it is concluded that breath samples may be stored in the bags for a 24-48 hr period, if necessary, prior t o analysis with negligible loss. The B H T has real potential for field use where immediate analysis following breath collection would be impossible. Large numbers of samples may be collected and run later in laboratory situations where the number of GC systems is limited. Subjects consumed only 0.25 g lactose as milk/kg body wt, the smallest dosage used with the breath HP test reported. I t is known that larger doses give severe symptoms in some of the subjects. T h e dosage given was adequate t o detect malabsorption, although individuals' mean response values ranged between 41.0 and 72.4 ppm. The times of maximum peak occurrence and the general overall appearance of response curves obtained are all very similar and resemble closely those obtained by Calloway and coworkers (18).Two subjects in this study reported only minor flatulence, otherwise no symptoms were experienced. The response on Day 1 was lowest in 4 of 5 subjects and lower than Day 2 in all 5 subjects. The level of breath HP generally rose on repeated exposure to lactose toward the third test day, then began to decline. However, data since collected on a large number of subjects (60) to be published later, do not necessarily show this same pattern. Also it has been hypothesized that H2-liberating fermentation might require periodic exposure of the intestinal flora to the appropriate sugar (6).However, three of the subjects reported here drank virtually no milk since childhood and two of them drank very little; yet their breath H P concentration always rose beyond 30 ppm when given approximately 15 g lactose. Thus, their nonconsumption of milk did not prevent a breath H2 response. Notable similarities were shown to exist between the B H T data on the malabsorber given a test diet with hydroTable IV. Lactose Malabsorption-Three Consecutive H, Concentrations after Skim Milk Consumption Sub-

ject

Day

Llinutes after lactose consumption t o maximum reading

3 Consecutive

readings, ppma

Tuesday 105 53, 57, 54 90 93, 90, 87 Thursday 76, 82, 82 Saturday 90 Monday 90 77, 85, 61 Wednesday 105 61, 67, 6 2 2 Tuesday 150 18, 30, 4 5 Thursday 44, 43, 55 150 68, 51, 51 Saturday 135 Monday 135 44, 51, 49 Wednesday 105 57, 61, 57 3 Tuesday 135 39, 40, 39 Thursday 135 44, 45, 4 1 39, 45, 57 Saturday 90 Monday 120 37, 33, 33 Wednesday 105 33, 45, 45 4 Tuesday 90 60, 53, 50 78, 72, 68 Thursday 105 59, 83, 52 Saturday 135 59, 58, 5 5 90 Monday 53, 52, 42 105 Wednesday 48, 17, 27 90 5 Tuesday 46, 39, 36 150 Thursday 54, 50, 48 90 Saturday 46, 44, 39 75 Monday 48, 49, 49 150 Wednesday a These three readings included the maximum hydrogen concentration.

ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976

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Table V. Mean Response Values for the Breath-H, Method Using Three Distinct Response Factors Maximum H, concentration 60-min

Average of 3 Highest H, Concentrations

Area

Zero-min corr.

Abs. value

60-min corr.

Zero-min corr.

Obsda

value

corrc

Zero-min cord

Tu Th S

5 5 5

M

5 5

50.0 63.4 69.2 55.6 55.0

37.4 50.0 57.4 55.0 42.2

40.8 52.4 61.4 48.8 48.8

42.0 58.7 59.9 51.4 51.8

29.2 44.8 47.8 39.9 39.2

32.8 47.7 52.1 44.6 45.6

3879 5423 5389 4764 4859

2383 2893 3873 3257 3348

2775 4098 4453 3962 4115

5 5 5 5 5

76.8 56.0 44.8 66.6 49.0

66.2 40.8 31.6 56.0 37.4

69.2 46.8 31.6 60.4 44.2

72.4 48.2 41.0 59.6 42.5

61.6 33.5 28.0 47.8 30.0

64.8 39.0 27.8 53.0 37.7

7205 3993 3646 5399 4071

5710 2463 2 11 4 3897 2569

6293 2892 2068 4655 3495

Source

Day

w

Abs. value

60-min corr.

In de 1

2 3 4 5 a

Number of observations. b Absolute value.

C

60-min correction. d Zero-min correction.

e

Subject identification number. _______ ~-

_________~

Table VI. Analysis of Variation for the Breath-H, Method Showing Mean Square Values Using Three Distinct Response Factors M.S. Max H , Con@

h1.S. Av of 3 Highest H, Concnsb

Zero-min Source

Day Indg Error

D.F.

Abs valued 60-min corre

289.1*i 855.7** 61.9 14%

293.2* 1019.5**

c0rr.f

277.88** 1072.8** 58.1 14%

Abs value

60-min corr.

Zero-min corr.

M.S. Area ( X 104)c

Abs value

60-min cow.

Zero-min corr.

290.4* 872.8** 55.4 15%

251.4* 258.2* 196.0* 188.91: 207.4* 1013.9**1060.2** 1079.7** 1099.1** 1354.1** 81.0 72.5 60.4 84.7 85.3 112.1 C.V.h 20% 22% 20% 18% 24% 24% a Mean squares of maximum H,concentration, b Mean squares of the average of the 3 highest consecutive H, concentration, C Mean squares of area under the curve. d Absolute value. e 60-min correction. f Zero-min correction. g Individual, h Coefficient o f variation. (Data marked denote F test significant at 5% level; data marked ** denote F test significant at 1%level. 4

4 16

*

lyzed lactose and that on absorbers given a test diet containing unhydrolyzed lactose where relatively flat curves were observed for all cases. I t should be noted, also, t h a t a small ( CD obtains, C; referring to the concentration of component i. Formal thermodynamic treatment of D partitioning between the gas phase and two immiscible, involatile, liquid phases yields the following GLC equation:

where K R is the observed infinite dilution partition coefficient of D between the “mixture” of A and S of volume fraction $A and the gas phase, and Kkr,k)and K&sj are the solute partition coefficients with pure A and pure S, re, with zero volume of mixspectively. Also, & = 1 - 4 ~and, ing, $t = C, V,, where V , is the molar volume of component i. With the additional assumption that the transfer of D between regions of A and S in the mixture is rapid (on the NMR time-scale), the following equation relating NMR (lhs) and GLC (rhs) quantities is derived:

where A() is the chemical shift difference (6: - 68), the zero superscript designating the shift in the pure liquid, and A is the difference (6 - a:), 6 being the measured shift a t volume fraction $A. Also, the chemical shifts refer t o those associated with a nucleus (generally a proton) of D. Equation 2 may be rearranged to give: (3)

where AK{ = K $ ( &-, KW,sj. T h e above equations would apply if “there is not random mixing in A + S mixtures, but rather a high degree of

ANALYTICAL CHEMISTRY, VOL. 48, NO. 2, FEBRUARY 1976

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