Butyrate Determination and Colon Cancer

By Jonathan V. Wright M.D.

As I walked through Meridian Valley Lab, one of the chemists called me over to his workbench. “Remember the stool butyrate determination you asked us to do for Mrs. S? Thought I’d let you know it’s the lowest level we’ve seen since we started testing for butyrate.” He showed me the numbers, and I made a mental note to start Mrs. S. on butyrate supplementation immediately, as well as the usual (but slower) manipulations of dietary fiber and/or intestinal microflora.

Mrs. S. had been in for a periodic examination. She noted no health problems except for persistent mild depression. However, she had another major concern: not dying of colon cancer. “My mother, my father, and two of my grandparents went that way,” she reported. “I’m doing everything I can not to go that way, too. I read Dennis Burkitt’s work on dietary fiber, and increased mine years ago. I’ve eliminated most sugar and food chemicals, though I could do better. I make sure to have a stool test done for blood periodically … it’s always been negative. Is there anything else I can do to prevent trouble?”

As part of her laboratory examinations, I’d made sure to have the level of butyrate in the stool checked. Considering her family history, it wasn’t much of a surprise that her results had set a lab-record low.

Butyrate

Butyrate serves as the most important energy source for normal colorectal epithelium. It also promotes the differentiation (progression towards normal) of cultured malignant cells. 1

Based on numerous studies, it appears highly probable that butyrate is a major cancer-inhibiting metabolite. Butyrate has been shown to “suppress the neoplastic state” in vitro of Syrian hamster cells. It induces differentiation in mouse leukemia cells, human promyelocytic leukemia cells, human retinoblastoma cells, and specific human breast carcinoma and colon carcinoma cell lines.2 According to these same authors: ” … when fiber is exposed to colonic flora, butyrate is the major metabolite. It has therefore been postulated that a possible mechanism for the anticancer action of fiber in the colon is due to increased fermentation of fiber to butyrate by the bacteria residing there.”

Fiber, Bacteria, and Butyrate

As a practitioner, I use the relatively inexpensive stool butyrate determination as an important “risk factor” test for colon cancer. Available evidence does not yet allow a precise calculation like the “cholesterol/VHDL ratio” atherosclerosis risk factor, but the evidence is strong enough to allow a general statement: “If the butyrate is lower, the colon cancer risk is higher.” Usually, stool butyrate values are easily altered by increasing or changing dietary fiber, and by manipulating the intestinal microflora. However, in difficult or urgent cases, or while waiting for the fiber and bacteria to sort themselves out properly, capsules of calcium/magnesium butyrate can also be used without significant hazard.

It should be noted that usually minor sources of substrate transformable to butyrate include undigested starch and colonic mucous.

Consumption of even “large” quantities of dietary fiber does not assure a normal stool butyrate level, as shown by the case of Mrs. S. The “proper” bacteria must be resident also. (Next ICNR will offer “ruminations” on human intestinal microflora.)

The stool butyrate test is inexpensive, therapy easy and natural, and the possibility of significant disease prevention very real … an ideal set of circumstances for the practitioner inclined towards disease prevention.

References:

1. Jass J.R., Diet Butyric Acid and Differentiation of Gastrointestinal Tract Tumors, Med. Hypoth 18, 113-118 (1985).
2. Anon, Effects of short-chain fatty acids on a human colon carcinoma cell line, Nutr Rev 46(1), 11-12 (1988).
3. Cummings, J.H. Short chain fatty acids in the human colon, Gut, 22, 763-779 (1981).

Effects of Short-Chain Fatty Acids on a Human Colon Carcinoma Cell Line

Butyrate slowed the growth rate and increased the production of alkaline phosphatase, dipeptidyl peptidase and carcinoembryonic antigen in a human colon carcinoma cell line.

Key Words: cancer, carcinoma cell line, human colon, short-chain fatty acids, butyrate, growth, carcinoembryonic antigen, dipeptidyl peptidase

Short-chain fatty acids are normally present in the lumen of the colon and serve as respiratory fuels for the colonic mucosa.1 Three short-chain fatty acids (Butyrate, acetate, and propionate) compose a major solute fraction of fecal water, and their principal source is from bacterial fermentation of nonabsorbed dietary carbohydrate such as fiber.1 Although their levels in stool can be modified by dietary modification, the average stool concentration of these fatty acids is approximately 190mM.

Butyrate is a major metabolite when fiber is broken down by colonic bacteria,1 and for several cell types butyrate has been shown to induce cell differentiation. Leavitt et al2 showed that in vitro butyric acid suppressed the neoplastic state of Syrian hamster cells. Butyrate also induces cell differentiation in the murine erythro-leukemia system, granulocyte differentiation in human promyelocytic leukemia cells, and cell differentiation of human retinoblastoma cells in a monolayer. Enhanced cell differentiation via butyrate has also been demonstrated for an antigen producing human breast carcinoma cell line MCF-7.3

Whitehead et al4 have established a colon carcinoma cell line LIM 1215, which was originally obtained from a 34 year-old man with metastatic colon cancer. The cell line is maintained in a medium containing fetal calf serum with variable amounts of hydrocortisone, insulin, a-thioglycerol, penicillin, and streptomycin. The authors5 studied the effects of various short- and medium-chain fatty acids on the growth and differentiation of this cell line. They assessed cell differentiation by measuring the alkaline phosphatase activity, dipeptidyl peptidase IV, carcinoembryonic antigen (CEA) concentrations, and mucin staining, all of which are indicators of cell differentiation. The cells were treated with trypsin and then resuspended in a medium at a concentration of 105 cells per ml. One ml aliquots of the cell suspension were put in well plates. Different dilutions of the short-chain fatty acids were then added to the wells, and the plates were incubated at 37C in an atmosphere of CO2 for up to 7 days. After incubation, the cells were trypsinized again until all the cells were detached from the growth surface. The total cell count per well was determined using a hemocytometer, and viability of the cells was determined by dye exclusion. Scanning and election microscopic studies were also done.

Growth studies showed that butyrate at concentrations of 1 mM and 10 mM significantly suppressed the growth of neoplastic cells. Propionate, however, only suppressed the growth of cells at 10 mM. After incubation with 1 mM butyrate, the number of cells per well was 2.0 + 0.2 x 105 in comparison to 8.1 + 1.2 x 105 cells in the control levels. Further studies with butyrate showed that 1 mM butyrate increased the doubling time from 26 hours in control culture to 72 hours in butyrate-treated cultures. This effect was not caused by toxicity since the viability of the cells was not affected and dead cells were not shed into the medium. There was a change in the morphology of cells within 2 to 3 days of incubation, that is, with butyrate the cells became larger and flatter.

The authors assessed the cloning efficiency of cells with and without 1 mM butyrate in the medium. This was judged by the ability of the cells to form colonies. In the control group, an inoculum of 103 cells produced 11 + 1 colonies, giving a cloning efficiency of 1.1 percent. In contrast, a much larger inoculum of 104 cells with butyrate produced only 5 + 3 colonies, giving a cloning efficiency of 0.05 percent.

One mM butyrate in the medium significantly increased the activity of alkaline phosphatase 600 percent over control. CEA production continued to increase for the first 4 days during the period of incubation with butyrate and then fell back toward control values. CEA is produced much more by colon tumors than by the normal colon. In tissue cultures, however, well-differentiated tumors produce more CEA than anaplastic tumors. One mM butyrate also increased the activity of the enzyme dipeptidyl amino peptidase. Histochemical studies confirmed that there were increased amounts of dipeptidyl peptidase IV. Scanning and electron microscope studies did not show any change in the number of distribution of microvilli in the cells incubated with butyrate.

These findings show that butyrate, which is a differentiating agent in other cell lines, has the same effect on a colon cancer cell line. The observations are interesting because when fiber is exposed to colonic flora, butyrate is the major metabolite. It has, therefore, been postulated that a possible mechanism for the anticancer action of fiber in the colon is due to increased fermentation of fiber to butyrate by the bacteria residing there.

References:
I. J.H. Cummings: Short-Chain Fatty Acids in the Human Colon. Gut 22:763-779,1981.
2. J. Levitt J.C. Barrett B.D. Crawford. and pop Ts’o: Butyric Acid Suppresion of the ln Vitro Neoplastic State of Syrian Harnster Cells. Nature 271:262-265. 1978.
3. K.N. Prasad and PK Sinha: Effect of Sodium Butyrate on Mammalin Cells in Culture:A Review.ln Vitro l2: 125-132, 1976.
4. R.H. Whitehead, F.A. Macr e, DJB St. John. and J. Ma: A Colon Cancer Cell Line (LIM1215) Derived from a patient with Inherited Nonpolyposis Colorectal Cancer. JNCI 74:759-765. 1985.
5. R.H. Whitehead, G.P. Young, and P.S. Bhatal: Effects of Short-Chain Fatty Acids on a New Human Colon Carcinoma Cell Line (LIM1215). Gut 27:1457-1463.1986.