The Journal of International Medical Research 1995; 23: 48-55 Faecal Triglycerides and Fatty Acids in the Differential Diagnosis ofpancreatic Insufficiency and Intestinal Malabsorption in Patients with Low fat Intakes T NAKAMURA, K TAKEBE, Y TANDO, Y ARAI, N YAMADA, M ISHII, H KIKUCHI AND K IMAMURA The Third Department of Internal Medicine, Hirosaki University School of Medicine, Hirosaki-shi, Aomori, Japan To investigate possible parameters for the differential diagnosis of steatorrhoea in patients with low fat intakes, faecal specimens were analysed from 15 patients with steatorrhoea due to chronic pancreatitis and seven patients with steatorrhoea due to intestinal malabsorption. The fat intakes ofthe patients ranged from 30.1 to 60 g, less than the average in American and European patients. The group with pancreatic steatorrhoea showed a significantly lower faecal output than the group with intestinal steatorrhoea but the two groups did not differ significantly in their total faecal fat excretion or concentration. The percentage triglycerides and the molecular ratio of triglycerides to fatty acids in the faeces were significantly higher (P < 0.01) in the group with pancreatic steatorrhoea than in those with intestinal steatorrhoea. The molecular percentage ratio of triglycerides to fatty acids was 6.8 ± 2.2 for the chronic pancreatitis group and 2.4 ± 1.0 for the intestinal malabsorption group; while the respective faecal hydroxy fatty acid contents were 3.1 ± 3.6% and 10.1 ± 3.3% (means ± SDs). These latter two parameters appeared to be the most valuable for distinguishing the two forms of steatorrhoea. 48
Y Tando et al. Faecaltriglycerides and fatty acids KEY WORDS; PANCREATIC STEATORRHOEA; INTESTINAL STEATORRHOEA; FAECAL TRIGLYCERIDE TO FATTY ACID MOLECULAR RATIO; FAECAL HYDROXY FATTY ACIDS INTRODUCTION It has been reported that faecal fat concentration plays a significant role in differentiating steatorrhoea in patients suffering from pancreatic disorders of digestion and absorption from a seemingly similar condition caused by disorders of the small intestine.' - 3 Specifically, the faecal fat content in pancreatic steatorrhoea has been reported to exceed 9.5% (g/100 g faeces) in many cases." The sensitivity of this differential diagnostic approach, however, is low unless the patient consumes 100 g of dietary fat daily and the daily faecal fat excretion exceeds 20 g.2 In Japan daily fat consumption by healthy subjects is only around 60 g,' and is lower still for sufferers of chronic pancreatitis." It is not possible to insist that patients increase their fat intake to a level approaching western standards. To tackle this problem, a simple method of determining the faecal triglyceride to fatty acid ratio was devised and applied to the differential diagnosis of clinical cases. In addition, faecal fat concentrations and hydroxy fat acid contents were compared in patients with pancreatic steatorrhoea and intestinal steatorrhoea. PATIENTS AND METHODS PATIENTS The 22 patients enrolled in this study comprised seven patients with intestinal malabsorption syndrome due to short bowel syndrome/blind loop syndrome and 15 with pancreatic malabsorption caused by chronic pancreatitis. A dietary survey was conducted on these patients for 3 to 7 days (including the period when the faecal specimens were collected) and the total calorie consumption and fat intake were computed. The dietary fat intake of patients with pancreatic steatorrhoea (44.7 g) was significantly lower (P < 0.01) than that of patients with intestinal steatorrhoea (54.3 g). Serum albumin, total cholesterol and haemoglobin levels (as nutritional indices) were, however significantly higher (P < 0.05, P < 0.01, P < 0.05) in patients with pancreatic steatorrhoea than in those with intestinal steatorrhoea. FAECAL SAMPLING AND ANALYSIS Faecal samples were collected for three consecutive days. After recording the total weight of the faeces, the samples were homogenized in twice their volume of water, and part was frozen. As internal standards, heptadecanoic acid and tricaprin were added to 100 mg of the freeze-dried sample and the lipids were extracted in perfusion in chloroform - methanol (2:1, v/v) for 2 h under a nitrogen steam. Water was added to the extract for separation and the chloroform layer was allowed to evaporate. Part of the product was saponified using 6 N-KOH methanol (5:95, v/v); and the fatty acids were extracted under acidic condition using n-hexane - ether (4:1; v/v) and methylated. This fatty acid methyl ester was applied to a HR-SS-I0 (25 m x 0.25 mm) column for gasliquid chromatographic analysis (Hitachi, Gas Chromatograph 163) at 195 -c. The sample was trimethylsilylated and subjected to a second chromatographic analysis. The new peak that developed on the chromatograph was regarded as representing hydroxy fatty acids." The remaining chloroform layer 49
Y Tando et al. Faecal triglycerides and fatty acids was trimethylsilylated and subjected to gas liquid chromatographic analysis (OV-l, 10 m x 0.25 mm, heated from 180 to 350 C at a rate of 5 C/min., using a Shimazu Gas Chromatograph GC14A). The procedure is a modified version of the simultaneous analysis of serum fatty acids and glycerides proposed by Myher." It facilitates quantitative analyses of fatty acids, monoglycerides, diglycerides, and triglycerides in separate groups. The yield of faecal lipids ranged from 87 to 103.5% and coefficient of variance, from 4.23 to 8.02% (n = 10). The peaks that represented phospholipids (phosphatidylcholine) and diglycerides overlapped. The percentage of the former in the faeces was small,":" however, and this peak was used for quantitative analysis of diglycerides. STATISTICAL ANALYSIS The Mann-Whitney U-test was used for statistical analysis. Values are expressed as means ± SDs. TABLE 1 Clinical features ofpatients with pancreatic or intestinal steatorrhoea No. and sex of patients Age (years) Operations Body mass index (kg/m 2 ) Daily calorie intake (kcal) Daily dietary fat intake (g) Serum albumin (gil) Cholesterol (mg/i) Haemoglobin (gil) D-xylose absorption rate (%) Pancreatic steatorrhoea 15 male 54.3 ± 7.5 (43-68) distal pancreatectomy 1 cholecystectomy 2 pancreatoduodenectomy 2 pancreatojejunostomy 1 18.4±2.6 (14.5-24.4) 1674.1 ± 188.2 (1311-2000) 44.7 ± 8.0** (30.1-60) 40 ± 5* (26-48) 1449 ± 428** (710-2520) 129±16* (103-153) Intestinal steatorrhoea 5 male, 2 female 52.4 ± 22.2 (15-82) short bowel 2 blind loop 5 (jejunotransversostomy) 19.4±1.0 (17.6-20.9) 1985.7 ± 226.8 (1600-2300) 54.3 ± 7.9 (40-60) 32±8 (22-47) 984 ± 339 (510-1390) 99 ± 14 (91-121) 25.7±11.0 (10.4-37.4) Values are means ± 80s with the rangein parenthesis. * p < 0.05; **P < 0.01 compared with the intestinal steatorrhoea group. 50
Y Tando et al. Faecal triglycerides and fatty acids RESULTS Sequential changes in the ratio of triglycerides to total faecal fats were observed in two patients (Fig. 1). The faecal sample was homogenized in water immediately after excretion. A sample that had been freezedried immediately after homogenization was preserved in a refrigerator at 4 DC to determine the percentage triglycerides in the freeze-dried faeces after 3, 7, and 14 days. The percentage neutral fat was 25.3% immediately after excretion but decreased steadily in case 1 (to 22.2%, 14.9%, and 7.1 % after 3,7, and 14 days, respectively). A slight reduction was also observed in case 2: the percentage neutral fat was 24.1 % immediately after excretion but decreased to 21.5,19.6, and 21.3% after 3,7, and 14 days, respectively. The faecal samples were, therefore, homogenized, freeze-dried, and analysed immediately after excretion. The mean faecal outputs of 460 g/day in the intestinal malabsorption group and 245.9 g/day in the group with untreated chronic pancreatitis differed significantly (P < 0.01, Table 2). No significant differences between the two disease groups were noted in the quantities of fat excreted in the faeces, fat concentrations, and percentages of fat absorbed. Two patients with chronic pancreatitis had faecal fat concentrations that exceeded the upper threshold (7.4%) in the intestinal malabsorption group. For patients with steatorrhoea due to malabsorption by the small intestine the percentage offatty acids in faecal fat was 92.5%, while in pancreatic steatorrhoea it was significantly lower, (P < 0.01) at 81.7% (Table 2). On the other hand, the percentage of triglycerides in the faecal fat of the group with pancreatic steatorrhoea (15.2%) was significantly higher (P < 0.01) than that for the 30 tti o J '0 20 ~ E Ql E oo.g 10 s o>, 0> ~ o FIGURE 1 o 3 7 Duration of refrigeration (d~ys) Changes in the proportions of triglycerides in the faecal fat of two patients (case 1, e; case 2, 0), after refrigeration of the faeces for 3, 7 or 14 days. The faeces had been homogenized in water and then freeze-dried immediately after excretion, before refrigeration. 14 51
Faecal output, faecal fat and its components, and faecal hydroxy fatty acids of patients with pancreatic steatorrhoea or intestinal steatorrhoea Group and Faecal Faecal Fat Fat Fatty Triglycerides TG/FA Hydroxy no. of patients output fat absorption in faeces acids (%)a (mol %) fatty acids (gfday) (gfday) (% of intake) (%) (%)a (%)a Pancreatic steatorrhoea Ql (n = 15) 254.9 ± 93.2** 11.9±10.7 72.7±21.1 4.7 ± 2.1 81.7 ± 5.4** 15.2 ± 4.0** 6.8 ± 2.2** 3.1 ± 3.6** N Intestinal steatorrhoea (n = 7) 460.0 ± 251.9 17.8± 16.7 68.8 ± 26.9 4.2± 2.2 92.5 ± 2.8 6.2±2.6 2.4 ± 1.0 10.1 ± 3.3 Normal values" <5 1.5±2.1 TG/FA (mol%), molecular percentage ratio of triglycerides to fats. a% of faecal fat. "Normal values are cited from reference 6. p < 0.01 compared with the intestinal steatorrhoea group. >'!j ~... III = (IJ ::j... Cl ~()C _... t.< Q C"l.., ~..,.., ::r... Q l:l ~ ~ Cl (IJ l:l-cl... = Cl l:l- ~;' Q... f!j.~ (IJ III C"l f.., ~,,<>:'" Q ~El :::r c:: ~~ ;:a.~ ~~ ~e SO
Y Tando et al. Faecaltriglycerides and fatty acids group with intestinal steatorrhoea (6.2%). No differences between the two groups were noted in the percentages of mono- and diglycerides. The faecal molecular percentage ratio of triglycerides to fatty acids was 2.4% for steatorrhoea caused by intestinal malabsorption and 6.8% for pancreatic steatorrhoea, the latter being significantly higher (P< 0.01). The percentage of hydroxy fatty acids in the faecal fatty acid was significantly higher (P < 0.01) for the intestinal steatorrhoea group (10.1%) than that for the pancreatic steatorrhoea group (P < 0.01). DISCUSSION To differentiate the fatty acid and triglycerides in faecal fats, the method of Van de Kamer" to separate neutral lipids and fatty acids by lability (or resistance) or faecal fats to hydrolysis is reported. A separation procedure that combines a solvent method with column chromatography has also been reported." Thompson et al." separated faecal fats into fatty acids and triglyceride spots by a simplified thin-layer-chromatographic method. Methods using isotopes - for example, tests using 131I-oleic acid and 131 1_ triolein12,13 or two different isotope species (e.g. 14e-triolein and 3H-oleic aoid}" - are more effective than the above mentioned chemical analysis in differentiating intestinal steatorrhoea from pancreatic steatorrhoea. These methods were not adopted, however, because they are either too cumbersome or pose safety problems by using isotopes. The method of serum analysis reported by Myher was modified and adapted to our method of simultaneous analysis of fatty acids and glycerides in faecal samples. According to most reports.v" the phospholipids detected in the tests constitute minor components in the faeces. Although the pho- spholipids formed peaks that overlapped those of the diglycerides, it is thought that the phospholipid component can safely be ignored. In the present study the preservation of the faecal samples following excretion was considered to be a more important element in the chemical analysis. Thompson et al," reported that the neutral fat fraction of a faecal sample is rapidly reduced if the sample is left standing at ambient temperature. In the present study the faecal samples were placed in the refrigerator immediately after their excretion but their neutral fat fractions had, nevertheless, been reduced after 3 days. We believe that this is due to the action of pancreatic lipase excreted into the faeces." A method was thus adopted in which the excreted faeces were immediately homogenized and freeze-dried for preservation. Pancreatic steatorrhoea and intestinal steatorrhoea are readily distinguished by the percentages of fatty acids and triglycerides in the faecal fat. In the present study the molecular percentage ratio of triglycerides to fatty acids was significantly lower in intestinal steatorrhoea than in pancreatic steatorrhoea and there was no overlap. We believe that the difference between intestinal malabsorption and disorders of digestion and absorption due to pancreatic diseases lies in the insufficiency of pancreatic lipase. Hence the simultaneous analysis of faecal fats is an effective method of differentiating between pancreatic and intestinal steatorrhoea. It has been reported recently that the faecal fat concentration in patients with pancreatic steatorrhoea is higher than that in patients with intestinal steatorrhoea and that this is an effective means of differentiating between the two clinical entities.' - 3 It has been reported that if 100 g of fat is consumed, of which more than 20 g is excreted in the faeces, then the faecal fat concen- 53
Y Tando et at. Faecal triglycerides and fatty acids tration will exceed 9.5%. In such cases a diagnosis of pancreatic steatorrhoea is appropriate; the diagnostic sensitivity of this approach is 47% and the specificity is 92%.2 These reports were, however, based on American or European subjects who normally consume as much as 100 g of dietary fat daily.z,3,17 We do not believe that the above-mentioned criteria apply to differential diagnosis in Japanese patients, whose fat intakes are much lower. Faecal fat analysis, was, therefore, carried out in patients on their normal, low-fat diet (i.e. fat intake ranging from 30.1 to 60 g).5 It was found that the faecal fat concentration was less than 8% in all those with intestinal steatorrhoea and over 8% in only two of 15 patients with pancreatic steatorrhoea. These results support the conclusion that the data for intestinal and pancreatic steatorrhoea overlap and that faecal fat content is not an effective parameter for differential diagnosis of the two diseases in Japanese patients.":" The faecal hydroxy fatty acid content (the end products of hydrolysis of dietary neutral fat by pancreatic lipase and the transform- ation of the resultant oleic acid into mainly 10-hydroxystearic acid by bacterial hydroxylase of the intestinal bacterial flora),g,zo has been used to differentiate intestinal from pancreatic steatorrhoea. In the present study the faecal hydroxy fatty acid content was significantly higher in patients with intestinal steatorrhoea than in those with pancreatic steatorrhoea, and resembled that related to bacterial overgrowth in conditions such as diabetic diarrhoea." Pancreatic steatorrhoea is the result of insufficient pancreatic lipase secretion, while intestinal steatorrhoea is the result of insufficient absorption of the hydrolysates of neutral fat, namely fatty acids. The present results support the conclusion that the molecular ratio of faecal triglycerides to fatty acids and the percentage of hydroxy fatty acids in the faecal fat (rather than the amount and concentration of fat excreted in the faeces) are effective parameters for use in the differential diagnosis of these two clinical entities when dietary fat consumption is normally low (as in Japanese patients). REFERENCES 1 Pedersen NT, Halgreen H, Worning H: Estimation of the 3-day fecal fat excretion and fat concentration as a differential test of malabsorption and maldigestion. ScandJGastroenterol1987; 22: 91-96. 2 Bo-Linn GW, Fordtran JS: Fecal fat concentration in patients with steatorrhea. Gastroenterology 1984; 87: 319-322. 3 Roberts 1M, Poturich C, Wald A: Utility of fecal fat concentrations as screening test in pancreatic insufficiency. Dig Dis Sci 1986; 31: 1021-1024. 4 Nakamura T, Kikuchi H, Takebe K, et al: Faecal lipid excretion levels in normal Japanese females on an unrestricted diet and a fat-restricted diet measured by simultaneous analysis of faecal lipids. J Int Med Res 1992; 20: 461-466. 5 Nakamura T, Arai Y, Terada A, et al: Dietary analysis of Japanese patients with chronic pancreatitis in stable conditions. JGastroenterol1994; 29(6): (in press). 6 Nakamura T, Imamura K, Kasai F, et al: Fecal excretions of hydroxy fatty acid and bile acid in diabetic diarrheal patients. J Diabetic Complications 1993; 7: 8-11. 7 Myher JJ: Separation and determination 54
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