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Technical Briefs |
1
Clinical Research Laboratory, Instituti Clinici di Perfezionamento, 20122 Milan, Italy, and
2
Department of Pediatrics, University of Sassari, Sassari 07100, Italy;
a address correspondence to this author at: Clinica Mangiagalli-Lab. Instituto Ostetrico Ginecologico, Clinical Research Laboratory, Via Commenda 12, 20122-Milano, Italy
The steatocrit is a semiquantitative method for measurement of fecal fat content (1); a few studies have reported its clinical applicability in the semiquantitative assessment of the degree of steatorrhea in premature infants and in several pediatric conditions, including cystic fibrosis and acute and chronic diarrhea (2)(3). However, one of these studies reported considerable difficulties with this method and could not find any correlation between the steatocrit and fecal fat excretion, which in contrast was well correlated with other semiquantitative tests, such as the Sudan microscopic method (4). It is interesting to note that most of the studies that demonstrated a good correlation between fecal fat excretion and steatocrit were carried out in Italy, where olive oil represents the main dietary fat, whereas the study in which such a relationship was not found was carried out in England, where dietary fats are saturated fatty acids of animal origin. This suggests that differences in the diet may influence the steatocrit. Methodological inadequacies may also underlie the above-mentioned discrepancies. In fact, although the steatocrit is a rather simple method, its results may be influenced by a series of chemical and physical variables, including the thoroughness of homogenization, acid strength, and the heating effects of hematocrit centrifuges. Recently, acidification of fecal homogenate was shown (5)(6) to lead to much better fat extraction through centrifugation, which led to greater sensitivity than the classic method and better prediction of fat content as measured by a reference chemical method (7). To further evaluate the reliability of the acid steatocrit, we assessed in vitro the influence of different kinds of fat at different concentrations on the acid and classic steatocrit methods.
Fresh stools were obtained from eight healthy subjects with no evidence
of gastrointestinal diseases; we then performed classic and acid
steatocrits, using the classic method described by Phuapradit et al.
(1) and the acid method described by Tran et al.
(5). All experiments were carried out at room temperature.
For the classic test, feces samples were weighed and diluted with 2 mL
of water per gram of feces. Hand homogenization was carried out with a
Potter system. Small amounts of homogenized feces were aspirated into
75-µL hematocrit capillaries; the tubes were sealed at one end with
sealing wax and centrifuged horizontally for 15 min at
12 000g in a standard hematocrit centrifuge. After
centrifugation, the tubes were placed in a vertical position so that
the presence of different layers was clearly evident. There was always
a basal solid layer (S) and an intermediate liquid layer,
and there was sometimes an upper fatty layer (F). We
measured the length of the solid and that of the fatty layer (when
present), using a graduated magnifying lens. The steatocrit is the
percentage of the length of the fatty layer over the length of the
solid plus the fatty layer:
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In our eight healthy subjects, no upper fatty layer was detectable, and therefore the classic steatocrit was negative.
The method used for the acid steatocrit was exactly the same as that of the classic test, except that before aspiration of the homogenate into the capillary tube, 700 mL/L perchloric acid was added to each sample in a volume equal to one-fifth of the homogenate volume. In our eight healthy subjects, the acid steatocrit was also "negative" (<1%).
The samples were then pooled together and divided into 0.5-g fractions, which were weighed with an analytical electronic balance (A&D; Bicasa); classic and acid steatocrits were also determined on the pooled fecal homogenate, and the results were negative. After the homogenate was mixed for 30 s with a standard vortex-type mixer, measured volumes of maize oil, olive oil, or butter (which was melted previously in a 37 °C water bath) were added separately with an adjustable volume pipetter (Bicasa) to portions of each sample, which were mixed for another 60 s. We performed classic and acid steatocrits on nine different samples containing separately maize oil, olive oil, or butter at increasing volumes per 0.5 g of homogenized stool: 20 µL (group A, three samples), 80 µL (group B, three samples), and 150 µL (group C, three samples). Classic and acid steatocrits were determined 14 times in the same day to calculate the within-day CV and for 18 consecutive days for the between-day CV. Differences in the results of classic and acid steatocrits were evaluated by means of the paired Student t-test.
The precision of the test was improved considerably by the use of acid
(Table 1
). The within-day CVs were 4.7–32% for the classic test but
only 1.8–7.1% for the acid steatocrit (P <0.01); the
between-day CVs were 2.4–40% for the classic test and 1.4–6.3% for
the acid steatocrit (P <0.05). The mean results (obtained
in the between-day precision experiments) were higher with the acid
steatocrit than with the classic method for each type of fat added at
each of the three different concentrations (groups A, B, and C; Table 1
). The increase was not proportional to the concentration, but was
particularly notable for samples with added olive oil (47–256%). The
CVs for samples with added butter (7.2–40%) were higher than the CVs
of samples with added olive (2.4–21%) or maize (5.6–36%) oil. With
regard to samples with added maize oil, the improvement in the CVs for
the acid steatocrit (1.4–7.1%) was substantially lower than that
found for samples with added butter.
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The present study was carried out to evaluate in vitro how different kind of fats are extracted from fecal homogenate and the effect of acidification on extraction efficiency. Our results provide additional evidence that fecal acidification improves fat extraction and precision. Tran et al. (6) reported an increase in acid steatocrit values ranging between 50% and 3364%, with a substantial improvement in fat extraction. They found that the amount of fat in the fatty layer was greater after acidification of the fecal sample and that acid steatocrit results agreed with chemically measured fecal fat better than the classic method did. Tran et al. (5) speculated that fecal acidification would lead to conversion of the ionized fatty acid species into protonated species, which would improve separation into lipid and water layers during the centrifugation step. This may occur to a greater extent for saturated fatty acids, as compared with unsaturated acids. Our data indicate that the type of fat present in the sample affects the extent to which acidification improves fat extraction from fecal homogenates. The importance of acidification was particularly evident for samples with added butter, which showed a major improvement of CVs, both for the within-day and the between-day precision assays when the acid steatocrit was performed. These findings seem to indicate that saturated fats of animal origin are more efficiently extracted when the fecal homogenate is acidified, and may explain the results obtained by Walters et al. (4) with the classic steatocrit in England.
We conclude that in vitro steatocrit values are influenced differently according to the type of fat present in the fecal homogenate. The steatocrit may thus be influenced by dietary composition, and its reference interval may vary according to differences in diet. The influence of dietary fat composition on steatocrit results may be more fully assessed in vivo through dietary modifications, but this approach may be difficult to achieve. Acidification may limit the clinical impact of these shortcomings by improving fat extraction from fecal homogenates.
Footnotes
References
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